Drug-conjugates, conjugation methods, and uses thereof

ABSTRACT

In certain aspects, compounds and uses thereof are provided. In certain aspects, compound-conjugates and uses thereof are provided.

BACKGROUND

Cytotoxic agents can provide therapeutic benefits in the treatment ofvarious conditions, including various cancers. Accordingly, it isdesirable to provide cytotoxic agents with therapeutically usefulproperties, for example, as chemotherapies in cancer treatments.

Clinical uses of cytotoxic agents as chemotherapies usually develop drugresistances, and thus, drop their therapeutic efficacies. Accordingly,it is desirable to provide cytotoxic agents with improved drugresistance profiles. One possibility is to design cytotoxic compoundswith efflux pump resistances. Another possibility is to design cytotoxiccompounds potentially act on more than one targets.

Tubulins are a class of targets for anti-mitotic agents aschemotherapies alone or in combination with other chemotherapies or asactive agent-conjugates. Examples of Tubulin-Binding Agents include, butare not limited to, the following compounds:

Many enzymes, for example, proteasome, MMAP, FAP and uPA, are consideredas cancer therapy targets due to their involvements in cancerproliferations and metastases. Examples of Proteasome inhibitorsinclude, but are not limited to, the following compounds:

Example of FAP Inhibitors, not Limited to:

Examples of uPA Inhibitors, not Limited to:

Examples of MMP Inhibitors Disclosed in, not Limited to:

Compounds disclosed in Bioorganic & Medicinal Chemistry 15 (2007)2223-2268.

Compounds disclosed in Cancer Metastasis Rev (2006) 25: 115-136.

SUMMARY

Some embodiments provide compounds, methods of preparing compounds, anduses thereof.

Some embodiments provide compound-conjugates, methods of preparingcompound-conjugates, and uses thereof.

Some embodiments provide a compound having the structure of Formula I:

Some embodiments provide a compound having the structure of Formula

or a pharmaceutically acceptable salt thereof,

wherein

B is a moiety might have contribution to enzyme inhibition or effluxpump resistance;

R₁-R₈ are each independently selected from the group consisting of II(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally R₇ and R together with the atoms to whichthey are attached are a cyclic 5- to 7-membered ring;

Y is CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR, where Ris C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

In some embodiments, B may be CN, CHO, CH₂OH, CH₂F, CH₂CN, CH₂N₃, COOH,CO—N(R)OR, CO—N(R)CO—R, CO—CO—NHR, CO—N(R)—SO₂R, (CH₂)_(p)COOH,(CH₂)_(p)—CH(OH)—COOH, CO—(CH₂)_(p)—COOH, CH═CH—COOH, CO—CH═CH—COOH,CH═CH—CONHOH, CH═CH—CONH—SO₂R, CO—CH═CH—CONHOH, B(OH)₂,(CH₂)_(p)—B(OH)₂, PO(OH)₂, or (CH₂)_(p)—PO(OH)₂, —R—COOH, —R—CO—N(R)OR,—R—CO—NHR, —R—CO—N(R)—SO₂R, where each occurrence of R is independentlyselected from H (hydrogen), C₁-C₈ alkyl, C₃-C₈ cycloalkyl, substitutedC₁-C₈ alkyl, substituted C₃-C₈ cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, and NR^(E)R^(F), and p is 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the compound having the structure of Formula I hasthe structure of Formula Ib:

or a pharmaceutically acceptable salt thereof,

B is a moiety might have contribution to enzyme inhibition or effluxpump resistance;

R₁-R₈ are each independently selected from the group consisting of HI(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally R₇ and R₈ together with the atoms towhich they are attached are a cyclic 5- to 7-membered ring;

Z is —F, —SR, —N₃, —NRR, —ONHR, —OAc, or —OR, where each R isindependently H, C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

In some embodiments, B may be CN, CHO, CH₂OH, CH₂F, CH₂CN, CH₂N₃, COOH,CO—NHOH, CO—CO—NHR, CO—NH—SO₂R, (CH₂)_(n)COOH, (CH₂)_(n)—CH(OH)—COOH,CO—(CH₂)_(n)—COOH. CH═CH—COOH, CO—CH═CH—COOH, CH═CH—CONHOH,CO—CH═CH—CONHOH, B(OH)₂, (CH₂)_(n)—B(OH)₂, PO(OH)₂, or(CH₂)_(n)—PO(OH)₂, where R=C₁-C₈ alkyl or substituted C₁-C₈ alkyl, and nis 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Some embodiments provide a compound having the structure of Formula IIa:

or a pharmaceutically acceptable salt thereof,

wherein X is OR¹⁰, selected from, but not limited to, a group consistingof at least one hetero atom:

wherein, X is SO₂R¹⁰, selected from, but not limited to, a groupconsisting of at least one hetero atom:

R₁-R₁₀ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₃ together with the atoms to which they are attachedare a cyclic 5- to 7-membered ring, or optionally and respectively R₇,R₈ and R₉ together with the atoms to which they are attached are acyclic 5- to 7-membered ring;

X is a group consisting of at least one heteroatom;

Y is CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR, where Ris C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

In some embodiments, the dual active compound having the structure ofFormula I has the structure of Formula IIb:

or a pharmaceutically acceptable salt thereof,

wherein, n is 0 or 1; X is OR¹⁰, selected from, but not limited to, agroup consisting of at least one hetero atom:

wherein, n is 0 or 1; X is SO₂R¹⁰, selected from, but not limited to, agroup consisting of at least one hetero atom:

R₁-R₁₀ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally and respectively R₇, R₈ and R₉ togetherwith the atoms to which they are attached are a cyclic 5- to 7-memberedring;

X is a group of at least one heteroatom;

Z is —F, —SR, —N₃, —NRR, —ONHR, —OAc, or —OR, where each R isindependently H, C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

Also provided herein are the compounds described above conjugated to atargeting moiety with a linker. Also provided herein are the compoundsdescribed above with a linker.

Some embodiments provide a compound having the structure of Formula IV:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R¹-R⁸ are each independently selected from the group consisting        of H hydrogen), optionally substituted C₁-C₈ alkyl, optionally        substituted C₁-C₈ alkoxy, optionally substituted C₃-C₈        cycloalkyl, optionally substituted aryl, and optionally        substituted heteroaryl, or optionally R¹ and R² together with        the nitrogen to which they are attached are an optionally        substituted cyclic 5- to 7-membered ring, or optionally R¹ and        R³ together with the atoms to which they are attached are an        optionally substituted cyclic 5- to 7-membered ring, or        optionally R⁷ and R⁸ together with the atoms to which they are        attached are an optionally substituted cyclic 5- to 7-membered        ring, or optionally R¹ is R^(1A) or R^(1B);    -   R^(1A) comprises a targeting moiety;    -   R^(1B) is -L¹(CH₂)_(n)R^(C), -L¹O(CH₂)_(n)R^(C) or        —(CH₂)_(n)R^(C);    -   R^(C) is C₁-C₈ alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, and        heterocyclyl, each optionally substituted with one or more        R^(D), or optionally R^(C) comprises a targeting moiety;    -   each R^(D) is independently selected from the group consisting        of —OH, —N₃, halo, cyano, nitro, —(CH₂)_(n)NR^(E)R^(F),        —(CH₂)_(n)C(═O)NR^(E)R^(F), —O(CH₂)_(n)NR^(E)R^(F),        —O(CH₂)_(n)C(═O)NR^(E)R^(F), —O(CH₂)_(m)OC(═O)NR^(E)R^(F),        —NR^(G)C(═O)R^(H), —NR^(G)S(O)_(z)R^(H),        —O(CH₂)_(m)O(CH₂)_(m)R^(J), —O(CH₂)_(n)C(═O)R^(J),        —O(CH₂)_(n)R^(J), optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted        heterocyclyl, and optionally substituted —O—(C₁-C₈ alkyl);    -   each NR^(E)R^(F) is independently selected, wherein R^(E) and        R^(F) are each independently selected from hydrogen,        -[(L¹)_(s)(C(R^(2A))₂)_(r)(NR^(2A))_(s)(C(R^(2A)h)_(r)]-[L¹(C(R^(2A))₂)_(r)(NR^(2A))(C(R^(2A))₂)_(r)]-(L¹)_(s)-R^(J),        -[(L¹)_(s)(C(R^(2A))₂)_(r)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-(L¹)_(s)[(C(R^(2A))₂)_(r)O(C(R^(2A))₂)_(r)(L²)_(s)]_(s)-(L¹)_(s)-R^(J),        optionally substituted C₁₋₈ alkyl, optionally substituted C₃₋₈        cycloalkyl, optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl;    -   each R^(G) is independently hydrogen, optionally substituted        C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted aryl, optionally substituted heteroaryl, or        optionally substituted heterocyclyl;    -   each R^(H) is independently hydrogen, optionally substituted        C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted heterocyclyl, or —NR^(E)R^(F);    -   each R^(J) is independently selected from the group consisting        of hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈        cycloalkyl, optionally substituted aryl, optionally substituted        heteroaryl, optionally substituted heterocyclyl,        —(CH₂)_(m)OR^(2B), —O(CH₂)_(m)OR^(2B), —(CH₂)_(n)NR^(2B)R^(2B),        —C(R^(2A))₂NR^(2B)R^(2B), —(CH₂)_(n)C(═O)OR^(2B), and        —C(═O)NHR^(2B);    -   each R^(2A) is independently selected, wherein R^(2A) is        selected from the group consisting of hydrogen, halo, —OH,        optionally substituted C₁-C₈ alkyl, optionally substituted        —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈ cycloalkyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted heterocyclyl, —(CH₂)_(n)OR^(2B),        —(CH₂)_(n)NR^(2C)R^(2C), —C(═O)OR^(2B), —C(═O)NR^(2C)R^(2C), or        optionally two geminal R^(2A) and the carbon to which they are        attached are together an optionally substituted three- to        six-membered carbocyclic ring;    -   each R^(2B) is independently selected from the group consisting        of hydrogen, —OH, —(CH₂)_(n)C(═O)OH,        C(═O)(C(R^(2D))₂)_(n)L³R^(2E), optionally substituted C₁-C₈        alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted —O—(C₁-C₈ alkyl), optionally substituted aryl,        optionally substituted heteroaryl, and optionally substituted        heterocyclyl;    -   each NR^(2C)R^(2C) is independently selected, wherein each        R^(2C) is independently selected from the group consisting of        hydrogen, —OH, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted —O—(C₁-C₈        alkyl), optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl, or        optionally both R^(2C) together with the nitrogen to which they        are attached are an optionally substituted heterocyclyl;    -   each R^(2D) is independently selected from the group consisting        of hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted —O—(C₁-C₈        alkyl), optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl;    -   each R^(2E) is independently selected from the group consisting        of optionally substituted C₁-C₈ alkyl, optionally substituted        C₃-C₈ cycloalkyl, optionally substituted aryl, optionally        substituted heteroaryl, and optionally substituted heterocyclyl,        and —(CH₂)_(n)C(═O)OR^(2F);    -   each R^(2F) is independently selected from the group consisting        hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl,        optionally substituted heteroaryl, and optionally substituted        heterocyclyl;    -   each L¹ is independently selected from the group consisting of        —C(═O)—, —S(═O)—, —C(═S)—, —S(═O)₂—, —C(═O)O—, —C(═O)NR^(2A)—,        —S(═O)NR^(2A)—, —S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and        —C(CF₃)₂NR^(2A)—;    -   each L² is independently selected from the group consisting of        optionally substituted aryl, optionally substituted heteroaryl,        and optionally substituted heterocyclyl;    -   each L³ is independently selected from the group consisting of        —C(═O)—, —S(═O)—, —C(═S)—, —S(═O)₂—, —C(═O)O—, —C(═O)NR^(2A)—,        —S(═O)NR^(2A)—, —S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and        —C(CF₃)₂NR^(2A)—;    -   each m independently is 1 or 2;    -   each n independently is 0, 1, 2, 3, 4, 5, or 6;    -   each r independently is 0, 1, 2, 3, 4, 5, or 6;    -   each s independently is 0 or 1; and    -   each z independently is 1 or 2    -   R⁷ is selected from the group consisting of H (hydrogen),        optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈        cycloalkyl, optionally substituted aryl, and optionally        substituted heterocyclyl;    -   R⁸ is selected from the group consisting of H (hydrogen),        —(CH₂)_(n)R^(C), optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl, and        optionally substituted heterocyclyl;    -   E is selected from the group consisting of:

-   -   where Y is CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or        CHOR¹⁴, where R¹⁴ is optionally substituted C₁-C₈ alkyl, and Z        is —F, —SR¹⁴, —N₃, —NR¹⁴R¹⁴, —ONHR¹⁴, —OAc, or —OR¹⁴, where each        R¹⁴ is independently H (hydrogen), C₁-C₈ alkyl substituted C₁-C₈        alkyl, or NR^(E)R^(F);

R¹¹-R¹³ are each independently selected from the group consisting of H(hydrogen), optionally substituted C₁-C₈ alkyl, optionally substitutedC₁-C₈ alkoxy, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; and

-   -   J is selected from: CN, CHO, CH₂OH, CH₂F, CH₂CN, CH₂N₃, COOH,        CO—N(R¹⁵)OR¹⁵, CO—N(R¹⁵)CO—R¹⁵, CO—CO—NHR¹⁵, CO—N(R¹⁵)—SO₂R¹⁵,        (CH₂)_(p)COOH, (CH₂)_(p)—CH(OH)—COOH, CO—(CH₂)_(p)—COOH,        CH═CH—COOH, CO—CH═CH—COOH, CH═CH—CONHOH, CH═CH—CONH—SO₂R,        CO—CH═CH—CONHOH, B(OH)₂, (CH₂)_(p)—B(OH)₂, PO(OH)₂, or        (CH₂)_(p)—PO(OH)₂, —R¹⁵—COOH, —CO—N(R¹⁵)OR¹⁵, —CO—N(R¹⁵)OR¹⁵,        —R¹⁵—CO—N(R¹⁵)OR¹⁵, —R¹⁵—CO—NHR¹⁵, —R¹⁵—CO—N(R¹⁵)—SO₂R¹⁵, where        each occurrence of R¹⁵ is independently selected from H        (hydrogen), C₁-C₈ alkyl, C₃-C₈ cycloalkyl, substituted C₁-C₈        alkyl, substituted C₃-C₈ cycloalkyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl and NR^(E)R^(F), and p is 0,        1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-10 shows the cytotoxic effects of compounds or antibody drugconjugates (ADCs) on various cell types. The antibody used in theantibody drug conjugates is Trastuzumab.

DETAILED DESCRIPTION

Some embodiments provide a compound.

In some embodiments, the compound includes a linker.

In some embodiments, the compound includes a cytotoxic agent.

In some embodiments, the compound includes a functional group that hastubulin-binding properties or tubulin inhibitory properties.

In some embodiments, the compound includes a functional group that hasprotease inhibitory or efflux pump resistant properties. For example,the functional group may have proteasome inhibitory properties.

Definitions

As used herein, common organic abbreviations are defined as follows:

-   Ac Acetyl-   aq. Aqueous-   BOC or Boc tert-Butoxycarbonyl-   BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate-   Bu n-Butyl-   ° C. Temperature in degrees Centigrade-   DCM methylene chloride-   DEPC Diethylcyanophosphonate-   DIC diisopropylcarbodiimide-   DIEA Diisopropylethylamine-   DMF N,N-Dimethylformamide-   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-   Et Ethyl-   EtOAc Ethyl acetate-   Eq Equivalents-   Fmoc 9-Fluorenylmethoxycarbonyl-   g Gram(s)-   h Hour (hours)-   HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium    hexafluorophosphate-   HOAt 1-Hydroxy-7-azabenzotriazole-   HOBT N-Hydroxybenzotriazole-   HOSu N-Hydroxysuccinimide-   HPLC High-performance liquid chromatography-   LC/MS Liquid chromatography-mass spectrometry-   Me Methyl-   MeOH Methanol-   MeCN Acetonitrile-   mL Milliliter(s)-   MS mass spectrometry-   RP-HPLC reverse phase HPLC-   rt room temperature-   t-Bu tert-Butyl-   TEA Triethylamine-   Tert, t tertiary-   TFA Trifluoracetic acid-   THF Tetrahydrofuran-   THP Tetrahydropyranyl-   TLC Thin-layer chromatography-   μL Microliter(s)

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of a compound and, which arenot biologically or otherwise undesirable for use in a pharmaceutical.In many cases, the compounds disclosed herein are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptableacid addition salts can be formed with inorganic acids and organicacids. Inorganic acids from which salts can be derived include, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylaminc, tripropylamine, andethanolamine. Many such salts are known in the art, as described in WO87/05297, Johnston et al., published Sep. 11, 1987 (incorporated byreference herein in its entirety).

As used herein, “C_(a) to C_(b)” or “C_(a-b)” in which “a” and “b” areintegers refer to the number of carbon atoms in the specified group.That is, the group can contain from “a” to “b”, inclusive, carbon atoms.Thus, for example, a “C₁ to C₄ alkyl” or “C₁₋₄ alkyl” group refers toall alkyl groups having from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, (CH)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkylthio” refers to the formula —SR wherein R is analkyl as is defined above, such as “C₁₋₉ alkylthio” and the like,including but not limited to methylmercapto, ethylmercapto,n-propylmercapto, 1-methylethylmercapto (isopropylmercapto),n-butylmercapto, iso-butylmercapto, sec-butylmercapto,tert-butylmercapto, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₋₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryloxy” and “arylthio” refers to RO— and RS—, in whichR is an aryl as is defined above, such as “C₆₋₁₀ aryloxy” or “C₆₋₁₀arylthio” and the like, including but not limited to phenyloxy.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₄alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃₋₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

A “(carbocyclyl)alkyl” is a carbocyclyl group connected, as asubstituent, via an alkylene group, such as “C₄₋₁₀ (carbocyclyl)alkyl”and the like, including but not limited to, cyclopropylmethyl,cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl,cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl,cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl, and the like. Insome cases, the alkylene group is a lower alkylene group.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkenyl” means a carbocyclyl ring or ring systemhaving at least one double bond, wherein no ring in the ring system isaromatic. An example is cyclohexenyl.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O, N or S, and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O, N, or S. Examplesof heterocyclyl rings include, but are not limited to, azepinyl,acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

A “(heterocyclyl)alkyl” is a heterocyclyl group connected, as asubstituent, via an alkylene group. Examples include, but are notlimited to, imidazolinylmethyl and indolinylethyl.

As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, andacryl.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

A “cyano” group refers to a “—CN” group.

A “cyanato” group refers to an “—OCN” group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—SCN” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “sulfinyl” group refers to an “—S(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alklyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 memberedheterocyclyl, as defined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-carbamyl” group refers to an “—N(R_(A))C(═O)OR_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-thiocarbamyl” group refers to a “—OC(═S)NR_(A)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl. C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “urea” group refers to a “—N(R_(A))C(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-thiocarbamyl” group refers to an “—N(R_(A))C(═S)OR_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂₋₈ alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substitutents independently selected fromC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O). Wherever a group isdescribed as “optionally substituted” that group can be substituted withthe above substituents.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

When two R groups are said to form a ring (e.g., a carbocyclyl,heterocyclyl, aryl, or heteroaryl ring) “together with the atom to whichthey are attached,” it is meant that the collective unit of the atom andthe two R groups are the recited ring. The ring is not otherwise limitedby the definition of each R group when taken individually. For example,when the following substructure is present:

and R¹ and R² are defined as selected from the group consisting ofhydrogen and alkyl, or R¹ and R² together with the nitrogen to whichthey are attached form a heterocyclyl, it is meant that R¹ and R² can beselected from hydrogen or alkyl, or alternatively, the substructure hasstructure:

where ring E is a heteroaryl ring containing the depicted nitrogen.

Similarly, when two “adjacent” R groups are said to form a ring“together with the atom to which they are attached,” it is meant thatthe collective unit of the atoms, intervening bonds, and the two Rgroups are the recited ring. For example, when the followingsubstructure is present:

and R¹ and R² are defined as selected from the group consisting ofhydrogen and alkyl, or R¹ and R² together with the atoms to which theyare attached form an aryl or carbocylyl, it is meant that R¹ and R² canbe selected from hydrogen or alkyl, or alternatively, the substructurehas structure:

where E is an aryl ring or a carbocylyl containing the depicted doublebond.

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. Thus, for example, a substituent depicted as -AE-or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

Compounds

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures, even when kinetically; theartisan recognizes that such structures may only represent a very smallportion of a sample of such compound(s). Such compounds are consideredwithin the scope of the structures depicted, though such resonance formsor tautomers are not represented herein.

Isotopes may be present in the compounds described. Each chemicalelement present in a compound either specifically or genericallydescribed herein may include any isotope of said element. For example,in a compound specifically or generically described herein a hydrogenatom may be explicitly disclosed or understood to be present in thecompound and each such hydrogen atom is any isotope of hydrogen,including but not limited to hydrogen-1 (protium) and hydrogen-2(deuterium). Thus, reference herein to a compound encompasses allpotential isotopic forms unless the context clearly dictates otherwise.

Utilities and Applications

Some embodiments provide a method of treating a patient in need thereofcomprising administering a compound as disclosed and described herein tosaid patient. In some embodiments, the patient may have cancer, aninfection, or an immune system disease. In some embodiments, thecompound may have anti-tumor, antibiotic, or anti-inflammatory activity.

Structures

Some embodiments provide a compound having the structure

or a pharmaceutically acceptable salt thereof, wherein: A is a tubulinbinding moiety; B is a group might have protease inhibitory or effluxpump resistant properties; and R₁-R₈ are each independently selectedfrom the group consisting of H (hydrogen), C₁-C₈ alkyl, substituted orcyclic C₁-C₈ alkyl, aryl, and substituted aryl, or optionally R₁ and R₂together with the nitrogen to which they are attached are a cyclic 5- to7-membered ring, or optionally R₁ and R, together with the atoms towhich they are attached are a cyclic 5- to 7-membered ring, oroptionally R₇ and R₈ together with the atoms to which they are attachedare a cyclic 5- to 7-membered ring. In some embodiments, the compoundmay be used alone as API (active pharmaceutical ingredient) or in aprodrug form. In some embodiments, the compound may be included in aconjugate including a linker and another component. In some embodiments,the compound may be included in a conjugate including a linker and atargeting moiety, such as antibody, Fab, peptide, protein ligand, andthe like. In some embodiments, the compound may be included in aconjugate including a linker and a carrier molecule, such as HSA, lipid,polymers, nanoparticles, and the like. In some embodiments, the compoundmay be included in a conjugate including a linker and a small moleculedrug/ligand, such as folic acid.

As used herein, the term “tubulin binding moiety” refers to a structuralcomponent of a compound that inhibits tubulin polymerization under acertain set of conditions. In some embodiments, the compound may inhibittubulin polymerization under in vivo or in vitro conditions. Forexample, the compound may inhibit tubulin polymerization in PBS.Examples of compounds that inhibit tubulin polymerization are describedin Peltier, et al., “The Total Synthesis of Tubulysin D,” J. Am. Chem.Soc., 2006, 128 (50): 16018-16019, and U.S. Publication No.:2005/0239713 the disclosures of which are incorporated herein byreference in their entirety.

As used herein, the term “functional moiety” refers to a structuralcomponent of a compound that interacts with a biological moiety orfragment of the biological moiety under a certain set of conditions. Insome embodiments, the functional moiety may interact with a biologicalmoiety or fragment of the biological moiety under in vivo or in vitroconditions. For example, the functional moiety may interact with abiological moiety or fragment of the biological moiety in PBS. In someembodiments, the functional moiety may afford a desirable effect in acompound comparison to a compound that does not include the functionalmoiety. In some embodiments, the functional moiety may contribute toenzyme inhibition or efflux pump resistance in a compound.

In some embodiments, the cytotoxic compound having the structure ofFormula Ia:

or a pharmaceutically acceptable salt thereof,

wherein,

B is a moiety might have contribution to enzyme inhibition or effluxpump resistance;

R₁-R₈ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally R₇ and R₈ together with the atoms towhich they are attached are a cyclic 5- to 7-membered ring;

Y is CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR, where Ris C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

In some embodiments, B may be CN, CHO, CH₂OH, CH₂F, CH₂CN, CH₂N₃, COOH,CO—NHOH, CO—CO—NHR, CO—NH—SO₂R, (CH₂)_(n)COOH, (CH₂)_(n)—CH(OH)—COOH,CO—(CH₂)_(n)—COOH, CH═CH—COOH, CO—CH═CH—COOH, CH═CH—CONHOH,CO—CH═CH—CONHOH, B(OH)₂, (CH₂)_(n)—B(OH)₂, PO(OH)₂, or(CH₂)_(n)—PO(OH)₂, where R is C₁-C₈ alkyl or substituted C₁-C₈ alkyl,and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the dual active compound having the structure ofFormula I has the structure of Formula Ib:

or a pharmaceutically acceptable salt thereof.

B is a moiety might have contribution to enzyme inhibition or effluxpump resistance;

R₁-R₈ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally R₇ and R₈ together with the atoms towhich they are attached are a cyclic 5- to 7-membered ring;

Z is —F, —SR, —N₃, —NRR, —ONHR, —OAc, or —OR, where each R isindependently H, C₁-C₈ alkyl or substituted C₁-C₈ alkyl

In some embodiments, B may be CN, CHO, CH₂OH, CH₂F, CH₂CN, CH₂N₃, COOH,CO—NHOH, CO—CO—NHR, CO—NH—SO₂R, (CH₂)_(n)COOH, (CH₂)_(n)—CH(OH)—COOH,CO—(CH₂)_(n)—COOH, CH═CH—COOH, CO—CH═CH—COOH, CH═CH—CONHOH,CO—CH═CH—CONHOH, B(OH)₂, (CH₂)_(n)—B(OH)₂, PO(OH)₂, or(CH₂)_(n)—PO(OH)₂, where R=C₁-C₈ alkyl or substituted C₁-C₈ alkyl, and nis 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Some embodiments provide a compound having the structure of Formula ha:

or a pharmaceutically acceptable salt thereof,

wherein X is OR¹⁰, selected from, but not limited to, a group consistingof at least one hetero atom:

wherein, X is SO₂R, selected from, but not limited to, a groupconsisting of at least one hetero atom:

R₁-R₁₀ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₃ together with the atoms to which they are attachedare a cyclic 5- to 7-membered ring, or optionally and respectively R₇,R₈ and R₉ together with the atoms to which they are attached are acyclic 5- to 7-membered ring;

X is a group consisting of at least one heteroatom;

Y is CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR, where Ris C₁-C₈ alkyl or substituted C₁-C₈ alkyl.

In some embodiments, the dual active compound having the structure ofFormula I has the structure of Formula IIb:

or a pharmaceutically acceptable salt thereof,

wherein, n is 0 or 1; X is OR¹⁰, selected from, but not limited to, agroup consisting of at least one hetero atom:

wherein, n is 0 or 1; X is SO₂R¹⁰, selected from, but not limited to, agroup consisting of at least one hetero atom:

R₁-R₁₀ are each independently selected from the group consisting of H(hydrogen), C₁-C₈ alkyl, and substituted or cyclic C₁-C₈ alkyl, oroptionally R₁ and R₂ together with the nitrogen to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₁ and R₃together with the atoms to which they are attached are a cyclic 5- to7-membered ring, or optionally and respectively R₇, R₈ and R₉ togetherwith the atoms to which they are attached are a cyclic 5- to 7-memberedring;

X is a group of at least one heteroatom;

Z is —F, —SR, —N₃, —NRR, —ONHR, —OAc, or —OR, where each R isindependently H, C₁-C₈ alkyl or substituted C₁-C₈ alkyl

Examples of compounds having the structure of Formula Ia include but notlimited to the following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of general compounds having the structure of Formula Ia includethe following:

Examples of compounds having the structure of Formula Ia include thefollowing:

Examples of compounds having the structure of Formula Ib include thefollowing:

Examples of compounds having the structure of Formula Ib include thefollowing:

In some embodiments, the compound is conjugated to a targeting moiety.

In some embodiments, the targeting moiety includes a monoclonal antibody(mAB). In some embodiments, the compound includes a spacer or amultifunctional linker.

In some embodiments, the spacer connects to the mAB by a group includinga N (nitrogen) atom. In some embodiments, the multifunctional linkerconnects to the mAB by a group including a N (nitrogen) atom. In someembodiments, the spacer or multifunctional linker may be optionallyconnected to an auxiliary moiety. In some embodiments, the auxiliarymoiety may be a second targeting moiety such as mAB and peptide. In someembodiments, the auxiliary moiety may be a hydrophilic polymer such aspolyethylene glycol (PEG), and the like. In some embodiments, the spaceror multifunctional linker may include a group including a N (nitrogen)atom. In some embodiments, the spacer or multifunctional linker mayinclude a cyclic group including a N (nitrogen) atom.

In some embodiments, the spacer connects to the mAB by a sulfide bond.In some embodiments, the multifunctional linker connects to the mAB by asulfide bond. In some embodiments, the spacer or multifunctional linkermay be optionally connected to an auxiliary moiety. In some embodiments,the auxiliary moiety may be a second targeting moiety such as mAB andpeptide. In some embodiments, the auxiliary moiety may be a hydrophilicpolymer such as polyethylene glycol (PEG), and the like. In someembodiments, the spacer or multifunctional linker may include a 2- to5-atom bridge. In some embodiments, the spacer or multifunctional linkermay include a 4C bridge.

Conjugation Methods, Spacers and Linkers Involved

Some embodiments provide a method of conjugating of a targeting moietythrough a spacer or a multifunctional linker.

In some embodiments, the spacer or multifunctional linker may include a2- to 5-atom bridge. In some embodiments, the method includes asingle-step or sequential conjugation approach. In some embodiments, thecompound includes a spacer or a multifunctional linker. In someembodiments, the spacer or multifunctional linker may include anoncleavable or cleavable unit such as peptides.

In some embodiments, the spacer or multifunctional linker may include agroup including a N (nitrogen) atom. In some embodiments, the methodincludes a single-step or sequential conjugation approach. In someembodiments, the spacer or multifunctional linker may include anoncleavable or cleavable unit such as a peptide.

Some embodiments provide a compound-conjugate having the structure ofFormula IIa:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   R¹-R⁹ are each independently selected from the group consisting        of H (hydrogen), optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl, and        optionally substituted heteroaryl, or optionally R¹ and R²        together with the nitrogen to which they are attached are an        optionally substituted cyclic 5- to 7-membered ring, or        optionally R¹ and R³ together with the atoms to which they are        attached are an optionally substituted cyclic 5- to 7-membered        ring, or optionally R⁷, R⁸ and R⁹ together with the atoms to        which they are attached are an optionally substituted cyclic 5-        to 7-membered ring, or optionally R¹ is R^(1A) or R^(1B);    -   R^(1A) comprises a targeting moiety;    -   R^(1B) is -L¹(CH₂)_(n)R^(C), -L¹O(CH₂)_(n)R^(C) or        —(CH₂)_(n)R^(C);    -   R^(C) is C₁-C₈ alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, and        heterocyclyl, each optionally substituted with one or more        R^(D), or optionally R^(C) comprises a targeting moiety;    -   each R^(D) is independently selected from the group consisting        of —OH, —N₃, halo, cyano, nitro, —(CH₂)_(n)NR^(E)R^(F),        —(CH₂)_(n)C(═O)NR^(E)R^(F), —O(CH₂)_(n)NR^(E)R^(F),        —O(CH₂)_(n)C(═O)NR^(E)R^(F), —O(CH₂)_(m)OC(═O)NR^(E)R^(F),        —NR^(G)C(═O)R^(H), —NR^(G)S(O)_(z)R^(H),        —O(CH₂)_(m)O(CH₂)_(m)R^(J), —O(CH₂)_(n)C(═O)R^(J),        —O(CH₂)_(m)R^(J), optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted        heterocyclyl, and optionally substituted —O—(C₁-C₈ alkyl);    -   each NR^(E)R^(F) is independently selected, wherein R^(E) and        R^(F) are each independently selected from hydrogen,        -[(L¹)_(s)(C(R^(2A))₂)_(r)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-[L¹(C(R^(2A))₂)_(r)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-(L¹)_(s)-R^(J),        -[(L¹)_(s)(C(R^(2A))₂)_(r)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-(L¹)_(s)[(C(R^(2A))₂)_(r)O(C(R^(2A))₂)_(r)(L²)_(s)]_(s)-(L¹)_(s)-R^(J),        optionally substituted C₁₋₈ alkyl, optionally substituted C₃₋₈        cycloalkyl, optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl;    -   each R^(G) is independently hydrogen, optionally substituted        C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted aryl, optionally substituted heteroaryl, or        optionally substituted heterocyclyl;    -   each R^(H) is independently hydrogen, optionally substituted        C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted heterocyclyl, or —NR^(E)R^(F);    -   each R^(J) is independently selected from the group consisting        of hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈        cycloalkyl, optionally substituted aryl, optionally substituted        heteroaryl, optionally substituted heterocyclyl,        —(CH₂)_(m)OR^(2B), —O(CH₂)_(m)OR^(2B), —(CH₂)NR^(2B)R^(2B),        —C(R^(2A))₂NR^(2B)R^(2B), —(CH₂)C(═O)OR^(2B), and        —C(═O)NHR^(2B);    -   each R^(2A) is independently selected, wherein R^(2A) is        selected from the group consisting of hydrogen, halo, —OH,        optionally substituted C₁-C₈ alkyl, optionally substituted        —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈ cycloalkyl,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted heterocyclyl, —(CH₂)_(m)OR^(2B),        —(CH₂)NR^(2C)R^(2C), —C(O)OR^(2B), —C(═O)NR^(2C)R^(2C), or        optionally two geminal R^(2A) and the carbon to which they are        attached are together an optionally substituted three- to        six-membered carbocyclic ring;    -   each R^(2B) is independently selected from the group consisting        of hydrogen, —OH, —(CH₂)_(n)C(═O)OH,        —C(═O)(C(R^(2D))₂))_(n)L³R^(2E), optionally substituted C₁-C₈        alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally        substituted —O—(C₁-C₈ alkyl), optionally substituted aryl,        optionally substituted heteroaryl, and optionally substituted        heterocyclyl;    -   each NR^(2C)R^(2C) is independently selected, wherein each        R^(2C) is independently selected from the group consisting of        hydrogen, —OH, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted —O—(C₁-C₈        alkyl), optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl, or        optionally both R^(2C) together with the nitrogen to which they        are attached are an optionally substituted heterocyclyl;    -   each R^(2D) is independently selected from the group consisting        of hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted —O—(C₁-C₈        alkyl), optionally substituted aryl, optionally substituted        heteroaryl, and optionally substituted heterocyclyl;    -   each R^(2E) is independently selected from the group consisting        of optionally substituted C₁-C₈ alkyl, optionally substituted        C₁-C₈ cycloalkyl, optionally substituted aryl, optionally        substituted heteroaryl, and optionally substituted heterocyclyl,        and —(CH₂)_(n)C(═O)OR^(2F);    -   each R^(2F) is independently selected from the group consisting        hydrogen, optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl,        optionally substituted heteroaryl, and optionally substituted        heterocyclyl;    -   each L¹ is independently selected from the group consisting of        —C(═O)—, —S(═O)—, —C(═S)—, —S(═O)₂—, —C(═O)O—, —C(═O)NR^(2A)—,        —S(═O)NR^(2A)—, —S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and        —C(CF₃)₂NR^(2A)—;    -   each L² is independently selected from the group consisting of        optionally substituted aryl, optionally substituted heteroaryl,        and optionally substituted heterocyclyl;    -   each L³ is independently selected from the group consisting of        —C(═O)—, —S(═O)—, —C(═S)—, —S(═O)₂—, —C(═O)O—, —C(═O)NR^(2A)—,        —S(═O)NR^(2A)—, —S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and        —C(CF₃)₂NR^(2A)—;    -   each m independently is 1 or 2;    -   each n independently is 0, 1, 2, 3, 4, 5, or 6;    -   each r independently is 0, 1, 2, 3, 4, 5, or 6;    -   each s independently is 0 or 1; and    -   each z independently is 1 or 2    -   R⁷ is selected from the group consisting of H (hydrogen),        optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈        cycloalkyl, optionally substituted aryl, and optionally        substituted heterocyclyl;    -   R⁸ is selected from the group consisting of H (hydrogen),        —(CH₂)_(n)R^(C), optionally substituted C₁-C₈ alkyl, optionally        substituted C₃-C₈ cycloalkyl, optionally substituted aryl, and        optionally substituted heterocyclyl;    -   X is a group consisting of at least one heteroatom, or selected        from groups consisting of —OR¹⁰, —SO2-R¹⁰, where R¹⁰ is R^(C).

In some embodiments, the active compound having the structure of FormulaI has the structure of Formula IIb:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R⁹ is selected from the group consisting of H (hydrogen),        optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈        cycloalkyl, substituted C₁-C₈ alkyl, substituted C₃-C₈        cycloalkyl, optionally substituted aryl, optionally substituted        heteroaryl, or optionally R⁷, R⁸ and R⁹ together with the atoms        to which they are attached are an optionally substituted cyclic        5- to 7-membered ring, X is selected from groups consisting of        —OR¹⁰, —SO2-R¹⁰, where R¹⁰ is R^(C); and n is 0 or 1.

In some embodiments, at least one of R¹, R¹⁰ and X comprises a targetingmoiety. In some embodiments, at least one of R¹, R¹⁰ and X furthercomprises a linker. In some embodiments, at least one of R¹, R¹⁰ and Xcomprises —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Insome embodiments, at least one of R¹, R¹⁰ and X comprises—(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, at least one of R¹, R¹⁰ and X comprises Val-Cit-PAB,Val-Ala-PAB, Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, orAla-Ala-Asn-PAB. In some embodiments, at least one of R¹, R¹⁰ and Xcomprises a peptide, oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—,Val-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB, D-Val-Lcu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, or combinations thereof. In some embodiments, thetargeting moiety is a monoclonal antibody (mAB). In some embodiments,the targeting moiety is an antibody fragment, surrogate, or variant. Insome embodiments, the targeting moiety is a protein ligand. In someembodiments, the targeting moiety is a protein scaffold. In someembodiments, the targeting moiety is a peptide. In some embodiments, thetargeting moiety is a small molecule ligand. In some embodiments, thelinker includes a 4-carbon bridge and at least two sulfur atoms. In someembodiments, the linker includes a fragment selected from the groupconsisting of:

In some embodiments, at least one of R¹, R¹⁰ and X comprises:

wherein:

-   -   the A-component is the targeting moiety; the E-component is an        optionally substituted heteroaryl or an optionally substituted        heterocyclyl; L³ is an optionally substituted C₁-C₆ alkyl, or L³        is null, when L³ is null the sulfur is directly connected to the        E-component; and L⁴ is an optionally substituted C₁-C₆ alkyl, or        L⁴ is null, when L⁴ is null the sulfur is directly connected to        the E-component. In some embodiments, the E-component includes a        fragment selected from the group consisting of:

In some embodiments, L³ is —(CH₂)—; and L⁴ is —(CH₂)—. In someembodiments, L³ is null; and L⁴ is null.

In some embodiments,

In some embodiments, at least one of R¹, R¹⁰ and X comprises:

wherein: A is the targeting moiety; X is N (nitrogen) or CH; Y is N(nitrogen), or CH; m is 0, 1, or 2; L is a linker, or null; and L^(1A)is a linker, or null. In some embodiments, L is null. In someembodiments, L includes —C(═O)—, —NH—C(═O)—, —C(═O)—O—, —NH—C(═O)—NH— or—NH—C(═O)—O—. In some embodiments, L is —C(═O)—, —NH—C(═O)—, —C(O)—O—,—NH—C(═O)—NH— or —NH—C(═O)—O—. In some embodiments, L is —C(═O)—. Insome embodiments,

In some embodiments, at least one of R¹, R¹⁰ and X comprises, consistsof, or consists essentially of:

wherein L⁵ may be optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈ cycloalkyl, optionally substituted C₁-C₈ alkoxy,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, or combination thereof. In someembodiments, L⁵ is an optionally substituted C₁-C₈ alkyl. In someembodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, at least one of R¹, R¹⁰ and X comprises, consistsof, or consist essentially of:

wherein A is the targeting moiety, L⁵ may be optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L⁵ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, at least one of R¹, R¹⁰ and X comprises, consistsof, or consist essentially of:

wherein A is the targeting moiety, L⁵ may be optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L⁵ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl. L⁶ maybe H, optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or combination thereof.

In some embodiments, at least one of R¹, R¹⁰ and X comprises, consistsof, or consist essentially of:

wherein A is the targeting moiety, L⁵ may be optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₃-C₈ cycloalkyl annulated to cyclooctyl ring, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L⁵ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl.

Some embodiments provide a compound-conjugate having the structure ofFormula V:

or a pharmaceutically acceptable salt thereof, wherein: A¹ may be atargeting moiety; B¹ is an auxiliary moiety that optionally includes asecond targeting moiety, or B¹ is null; L¹ includes a group including aN (nitrogen) atom or a group including a 2- to 5-carbon bridge and atleast one sulfur atom; each D is independently selected, where each Dincludes a compound; each L² is independently a linker, wherein at leastone L² links to L¹; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, A¹ may be a monoclonal antibody (mAB). In some embodiments,A¹ may be an antibody fragment, surrogate, or variant. In someembodiments, A¹ may be a protein ligand. In some embodiments, A¹ may bea protein scaffold. In some embodiments, A¹ may be a peptide. In someembodiments. A¹ may be RNA or DNA. In some embodiments, A¹ may be a RNAor DNA fragment. In some embodiments, A¹ may be a small molecule ligand.In some embodiments, B¹ may be a hydrophilic polymer. In someembodiments, the hydrophilic polymer may polyethylene glycol (PEG), andthe like. In some embodiments, B¹ may be a biodegradable polymer. Insome embodiments, the biodegradable polymer may be unstructured proteinspolyamino acids, polypeptides polysaccharides and combinations thereof.In some embodiments, B¹ may be a monoclonal antibody (mAB). In someembodiments, B¹ may be an antibody fragment, surrogate, or variant. Insome embodiments, B¹ may be a protein ligand. In some embodiments, B¹may be a protein scaffold. In some embodiments, B¹ may be a peptide. Insome embodiments, B¹ may be RNA or DNA. In some embodiments, B¹ may be aRNA or DNA fragment. In some embodiments, B¹ may be a small moleculeligand. In some embodiments, D may includes a biologically activecompound. In some embodiments, D may includes a core from tubulin-binderor tubulin-binder derivative. In some embodiments, D include a core fromepothilone A, epothilone B, paclitaxel, or derivatives thereof. In someembodiments, D includes

wherein: A is a tubulin binding moiety; B is a protease inhibitionmoiety; and R₁-R₈ are each independently selected from the groupconsisting of H (hydrogen), C₁-C₈ alkyl, substituted or cyclic C₁-C₈alkyl, aryl, and substituted aryl, or optionally R₁ and R₂ together withthe nitrogen to which they are attached are a cyclic 5- to 7-memberedring, or optionally R₁ and R₃ together with the atoms to which they areattached are a cyclic 5- to 7-membered ring, or optionally R₇ and R₈together with the atoms to which they are attached are a cyclic 5- to7-membered ring. In some embodiments, A may be

and Y may be CH₂, S, S═O, C═O, CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR,where R is C₁-C₈ alkyl or substituted C₁-C₈ alkyl. In some embodiments,A may be

Z may be N (nitrogen), CH, C—OH, C—OR, CSH, CSR, where R is C₁-C₈ alkylor substituted C₁-C₈ alkyl; X may be F, OH, N₃, OMe, or OR, where R isC₁-C₈ alkyl or substituted C₁-C₈ alkyl; and V may be CH₂, S, S═O, C═O,CHF, CHCN, CHN₃ CH—OH, CH—ONH₂, or CHOR, where R is C₁-C₈ alkyl orsubstituted C₁-C₈ alkyl. In some embodiments, L² may include a spacer ora multifunctional linker. In some embodiments, L² may include a spacerand a multifunctional linker. In some embodiments, L² may include amultifunctional linker. In some embodiments, each L² may be a linker,wherein the linker may be cleavable or non-cleavable under biologicalconditions. In some embodiments, the linker may be cleavable by anenzyme. In some embodiments, L² may include Linker. In some embodiments,L¹ includes a cyclic group including at least one N (nitrogen) atom. Insome embodiments, L¹ includes a cyclic group including at least two N(nitrogen) atoms. In some embodiments, L¹ includes a cyclic groupincluding at least one N (nitrogen) atom and a spacer. In someembodiments, L¹ includes a cyclic group including at least two N(nitrogen) atoms and a spacer. In some embodiments, the spacer connectsto the mAB by an amide bond. In some embodiments, the spacer connects tothe mAB through an amine bond. In some embodiments, L¹ includes a 2- to5-carbon bridge and at least one sulfur atom. In some embodiments, L¹includes a 2- to 5-carbon bridge and at least two sulfur atoms. In someembodiments, L¹ includes a 2- to 5-carbon bridge and a spacer. In someembodiments, L¹ includes a 2- to 5-carbon bridge, at least two sulfuratoms and a spacer. In some embodiments, L¹ may include one or moresulfurs. In some embodiments, the L¹ may include two or more sulfurs. Insome embodiments, the L¹ may include exactly two sulfurs. In someembodiments, may include a 4-carbon bridge and/or a spacer. In someembodiments, L¹ include a 4-carbon bridge or a spacer. In someembodiments, L¹ may include a 4-carbon bridge and a spacer. In someembodiments, L¹ includes a 4-carbon bridge and at least two sulfuratoms. In some embodiments, the spacer connects to the mAB by a sulfidebond. In some embodiments, the spacer connects to the mAB through athioether. In some embodiments, A¹ comprises at least one modifiedn-butyl L-α-amino acid. In some embodiments, at least one modifiedL-Lysine residue is from an L-Lysine residue of a peptide beforeconjugation. In some embodiments, at least one nitrogen of L¹ is from anat least one modified n-butyl L-α-amino acid of a peptide beforeconjugation. In some embodiments, A¹ and L¹ together comprise at leastone modified L-Lysine residue. In some embodiments, the terminalnitrogen of the side chain of an L-Lysine residue of a peptide beforeconjugation is the at least one N (nitrogen) atom of L¹. In someembodiments, A¹ comprises the —(CH₂)₄— of the side chain of an L-Lysineresidue of a peptide before conjugation that provides the at least one N(nitrogen) atom of L¹. In some embodiments, A¹ comprises a modifiedn-butyl α-amino acid residue. In some embodiments, Linker may be apeptide. In some embodiments, Linker may include an oligosaccharide. Forexample, Linker may include chitosan. In some embodiments, L² mayinclude Linker and —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. In some embodiments, L² may include Linker and —(CH₂CH₂O)_(n)— wheren is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Linker mayinclude —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, Linker may include —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In some embodiments, Linker may includeVal-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB, D-Val-Lcu-Lay, Gly-Gly-Arg,Ala-Ala-Asn-PAB, or the like. In some embodiments, Linker may includeany combination of peptide, oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)—,Val-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB, D-Val-Leu-Lay, Gly-Gly-Arg,Ala-Ala-Asn-PAB, and the like. In some embodiments, the spacer mayinclude a peptide. In some embodiments, the spacer may include anoligosaccharide. For example, the spacer may include chitosan. In someembodiments, the spacer may include —(CH₂)_(n)— where n is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In some embodiments, L¹ may include a componentincluding a 4-carbon bridge and —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In some embodiments, the spacer may include—(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, L¹ may include a component including a 4-carbon bridge and—(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, the spacer may include Val-Cit-PAB, Val-Ala-PAB,Phe-Lys-PAB, Ala-Ala-Asn-PAB, or the like. In some embodiments, thespacer may be any combination of peptide, oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB, Ala-Ala-Asn-PAB,and the like. In some embodiments, L¹ may include,

but is not limited to,

and the like. In some embodiments, L¹ may include, but is not limitedto,

and the like.

In some embodiments, the compound-conjugates may include one or morecomponents selected from the group consisting of an amino acid, an aminoacid residue, an amino acid analog, and a modified amino acid.

As used herein, the term “peptide” refers to a structure including oneor more components each individually selected from the group consistingof an amino acid, an amino acid residue, an amino acid analog, and amodified amino acid. The components are typically joined to each otherthrough an amide bond.

As used herein, the term “amino acid” includes naturally occurring aminoacids, a molecule having a nitrogen available for forming an amide bondand a carboxylic acid, a molecule of the general formula NH₂—CHR—COOH orthe residue within a peptide bearing the parent amino acid, where “R” isone of a number of different side chains. “R” can be a substituent foundin naturally occurring amino acids. “R” can also be a substituentreferring to one that is not of the naturally occurring amino acids.

As used herein, the term “amino acid residue” refers to the portion ofthe amino acid which remains after losing a water molecule when it isjoined to another amino acid.

As used herein, the term “amino acid analog” refers to a structuralderivative of an amino acid parent compound that often differs from itby a single element.

As used herein, the term “modified amino acid” refers to an amino acidbearing an “R” substituent that does not correspond to one of the twentygenetically coded amino acids.

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids are conventional and are as follows: TheD-amino acids are designated by lower case, e.g. D-proline=p, etc.

TABLE 1 Amino Acids One-Letter Symbol Common Abbreviation Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

Certain amino acid residues in the compound-conjugate can be replacedwith other amino acid residues without significantly deleteriouslyaffecting, and in many cases even enhancing, the activity of thepeptides. Thus, also contemplated by the preferred embodiments arealtered or mutated forms of the active agent-conjugate wherein at leastone defined amino acid residue in the structure is substituted withanother amino acid residue or derivative and/or analog thereof. It willbe recognized that in preferred embodiments, the amino acidsubstitutions are conservative, i.e., the replacing amino acid residuehas physical and chemical properties that are similar to the amino acidresidue being replaced.

For purposes of determining conservative amino acid substitutions, theamino acids can be conveniently classified into two maincategories—hydrophilic and hydrophobic—depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into nonpolar andaromatic amino acids. The definitions of the various categories of aminoacids are as follows:

The term “hydrophilic amino acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) andArg (R).

The term “hydrophobic amino acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:1.25-142. Genetically encoded hydrophobic amino acids include Pro(P), lie (1), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly(G) and Tyr (Y).

The term “acidic amino acid” refers to a hydrophilic amino acid having aside chain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

The term “basic amino acid” refers to a hydrophilic amino acid having aside chain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

The term “polar amino acid” refers to a hydrophilic amino acid having aside chain that is uncharged at physiological pH, but which has at leastone bond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

The term “nonpolar amino acid” refers to a hydrophobic amino acid havinga side chain that is uncharged at physiological pH and which has bondsin which the pair of electrons shared in common by two atoms isgenerally held equally by each of the two atoms (i.e., the side chain isnot polar). Genetically encoded nonpolar amino acids include Leu (L),Val (V), Ile (I), Met (M), Gly (G) and Ala (A).

The term “aromatic amino acid” refers to a hydrophobic amino acid with aside chain having at least one aromatic or heteroaromatic ring. In someembodiments, the aromatic or heteroaromatic ring may contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR andthe like where each R is independently (C₁-C₆) alkyl, substituted(C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

The term “aliphatic amino acid” refers to a hydrophobic amino acidhaving an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Lcu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfanyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with —SHcontaining side chains) to exist in a peptide in either the reduced free—SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg, 1984, supra), it is to beunderstood that for purposes of the preferred embodiments Cys (C) iscategorized as a polar hydrophilic amino acid, notwithstanding thegeneral classifications defined above.

As used herein, the term “targeting moiety” refers to a structure thatbinds or associates with a biological moiety or fragment thereof.

In some embodiments, the targeting moiety may be a monoclonal antibody(mAB). In some embodiments, the targeting moiety may be an antibodyfragment, surrogate, or variant. In some embodiments, the targetingmoiety may be a protein ligand. In some embodiments, the targetingmoiety may be a protein scaffold. In some embodiments, the targetingmoiety may be a peptide. In some embodiments, the targeting moiety maybe RNA or DNA. In some embodiments, the targeting moiety may be a RNA orDNA fragment. In some embodiments, the targeting moiety may be a smallmolecule ligand.

In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Janthur et al., “Drug Conjugates Such as Antibody DrugConjugates (ADCs), Immunotoxins and Immunoliposomes Challenge DailyClinical Practice,” Int. J. Mol. Sci. 2012, 13, 16020-16045, thedisclosure of which is incorporated herein by reference in its entirety.In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Trail, P A, “Antibody Drug Conjugates as CancerTherapeutics,” Antibodies 2013, 2, 113-129, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the targeting moiety may be HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.

In some embodiments, the targeting moiety may be Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of Brentuximab, Inotuzumab, Gemtuzumab, Milatuzumab,Trastuzumab, Glembatumomab, Lorvotuzumab, or Labestuzumab.

As used herein, the term “linker” refers to a moiety that connects twoor more components to each other.

In some embodiments, the linker may be a linker disclosed in Janthur etal., “Drug Conjugates Such as Antibody Drug Conjugates (ADCs),Immunotoxins and Immunoliposomes Challenge Daily Clinical Practice,”Int. J. Mol. Sci. 2012, 13, 16020-16045. In some embodiments, the linkermay be a linker disclosed in Trail, P A, “Antibody Drug Conjugates asCancer Therapeutics,” Antibodies 2013, 2, 113-129. In some embodiments,the linker may be a linker disclosed in U.S. Pat. No. 7,829,531.

In some embodiments, the linker may comprise, consist of, or consistessentially of the linker portion of HuM195-Ac-225, HuM195-Bi-213,Anyara (naptumomab estafenatox; ABR-217620), AS1409, Zevalin(ibritumomab tiuxetan), BIIB15, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Lcu16-IL2, SAR3419, SGN-35, or CMC544.

In some embodiments, the linker may comprise, consist of, or consistessentially of the linker portion of Brentuximab vedotin, Trastuzumabemtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine, Glembatumumabvedotin, SAR3419, Moxetumomab pasudotox, Moxetumomab pasudotox,AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, or IMGN-388.

In some embodiments, the linker may comprise, consist of, or consistessentially of Valine-citrulline residue, hydrazine, 4-mercaptobutanoyl,4-(N-succinimidomethyl)cyclohexanecarbonyl (SMCC), Maleimidocaproyl,Phenylalaninelysine, 6-(3-(thio)propanamido)hexanoyl,3-mercaptopropanoyl, 4-mercaptopentanoyl, or lysine residue.

In some embodiments, the linker may comprise, consist of, or consistessentially of:

wherein L⁵ may be optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈ cycloalkyl, optionally substituted C₁-C₈ alkoxy,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, or combination thereof. In someembodiments, L⁵ is an optionally substituted C₁-C₈ alkyl. In someembodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, the linker may comprise, consist of, or consistessentially of:

wherein A is the targeting moiety, L⁵ may be optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L⁵ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, the linker may comprise, consist of, or consistessentially of:

wherein A is the targeting moiety, L⁵ may be optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L⁵ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl. L⁶ maybe H, optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or combination thereof.

In some embodiments, the linker may comprise, consist of, or consistessentially of:

herein A is the targeting moiety, L⁵ may be optionally substituted C₁-C₈alkyl, optionally substituted C₃-C₈ cycloalkyl, optionally substitutedC₃-C₈ cycloalkyl annulated to cyclooctyl ring, optionally substitutedC₁-C₈ alkoxy, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, or combination thereof.In some embodiments, L⁵ is an optionally substituted C₁-C₈ alkyl. Insome embodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, the linker may comprise, consist of, or consistessentially of:

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the amino acid substitutions need notbe, and in certain embodiments preferably are not, restricted to thegenetically encoded amino acids. In some embodiments, the activeagent-conjugate may contain genetically non-encoded amino acids. Thus,in addition to the naturally occurring genetically encoded amino acids,amino acid residues in the active agent-conjugate may be substitutedwith naturally occurring non-encoded amino acids and synthetic aminoacids.

Certain commonly encountered amino acids which provide usefulsubstitutions for the active agent-conjugates include, but are notlimited to, β-alanine (β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ϵ-aminohexanoic acid(Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);omithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine(t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-phenylphenylalanine, 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu);2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH₂));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines).

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

The classifications of the genetically encoded and common non-encodedamino acids according to the categories defined above are summarized inTable 2, below. It is to be understood that Table 2 is for illustrativepurposes only and does not purport to be an exhaustive list of aminoacid residues and derivatives that can be used to substitute the activeagent-conjugate described herein.

TABLE 2 CLASSIFICATIONS OF COMMONLY ENCOUNTERED AMINO ACIDS GeneticallyClassification Encoded Non-Genetically Encoded Hydrophobic Aromatic F,Y, W Phg, Nal, Thi, Tic, Phe (4-Cl), Phe (2-F), Phe (3-F), Phe (4-F),hPhe Nonpolar L, V, I, M, G, t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, A, PMcGly, Aib Aliphatic A, V, L, I b-Ala, Dpr, Aib, Ahx, MeGly, t-BuA, t-BuG, MeIle, Cha, Nle, MeVal Hydrophilic Acidic D, E Basic H, K, R Dpr,Orn, hArg, Phe(p-NH₂), Dbu, Dab Polar C, Q, N, S, T Cit, AcLys, MSO,bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (in L-peptides)

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

While in most instances, the amino acids of the compound-conjugate willbe substituted with L-enantiomeric amino acids, the substitutions arenot limited to L-enantiomeric amino acids. In some embodiments, thepeptides may advantageously be composed of at least one D-enantiomericamino acid. Peptides containing such D-amino acids are thought to bemore stable to degradation in the oral cavity, gut or serum than arepeptides composed exclusively of L-amino acids.

Examples of compound-conjugates include, but are not limited to, thefollowing general compounds:

R may be H (hydrogen), C₁-C₈ alkyl, substituted or cyclic C₁-C₈ alkyl

X may be S (sulfur), CH₂, CH—OH, CH—OR, CH—ONH₂, or C═O.

Examples of compounds include, but are not limited to, the followinggeneral compounds:

Examples of compound-conjugates include, but are not limited to, thefollowing general compounds:

R may be H (hydrogen), C₁-C₈ alkyl, substituted or cyclic C₁-C₈ alkyl,and the like X may be S (sulfur), CH₂, CH—OH, CH—OR, CH—ONH₂, C═O, andthe like.

Examples of compounds include, but are not limited to, the followingcompound and general compounds:

Pharmaceutical Compositions

In some embodiments, the compounds disclosed herein are used inpharmaceutical compositions. The compounds can be used, for example, inpharmaceutical compositions comprising a pharmaceutically acceptablecarrier prepared for storage and subsequent administration. Also,embodiments relate to a pharmaceutically effective amount of theproducts and compounds disclosed above in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), which is incorporated herein by reference in itsentirety. Preservatives, stabilizers, dyes and even flavoring agents canbe provided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be addedas preservatives. In addition, antioxidants and suspending agents can beused.

The compositions can be formulated and used as tablets, capsules, orelixirs for oral administration; suppositories for rectaladministration; sterile solutions, suspensions for injectableadministration; patches for transdermal administration, and sub-dermaldeposits and the like. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), can be utilized.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions can be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions can be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

To formulate the compounds of Formulae I and II as an anti-cancer agent,known surface active agents, excipients, smoothing agents, suspensionagents and pharmaceutically acceptable film-forming substances andcoating assistants, and the like can be used. Preferably alcohols,esters, sulfated aliphatic alcohols, and the like can be used as surfaceactive agents; sucrose, glucose, lactose, starch, crystallizedcellulose, mannitol, light anhydrous silicate, magnesium aluminate,magnesium methasilicate aluminate, synthetic aluminum silicate, calciumcarbonate, sodium acid carbonate, calcium hydrogen phosphate, calciumcarboxymethyl cellulose, and the like can be used as excipients;magnesium stearate, talc, hardened oil and the like can be used assmoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soyacan be used as suspension agents or lubricants; cellulose acetatephthalate as a derivative of a carbohydrate such as cellulose or sugar,or methylacetate-methacrylate copolymer as a derivative of polyvinyl canbe used as suspension agents; and plasticizers such as ester phthalatesand the like can be used as suspension agents. In addition to theforegoing preferred ingredients, sweeteners, fragrances, colorants,preservatives and the like can be added to the administered formulationof the compound produced by the method of the embodiment, particularlywhen the compound is to be administered orally.

When used as an anti-cancer compound, for example, the compounds ofFormulae I and II or compositions including compounds of Formulae I andII can be administered by either oral or non-oral pathways. Whenadministered orally, it can be administered in capsule, tablet, granule,spray, syrup, or other such form. When administered non-orally, it canbe administered as an aqueous suspension, an oily preparation or thelike or as a drip, suppository, salve, ointment or the like, whenadministered via injection, subcutaneously, intraperitoneally,intravenously, intramuscularly, or the like.

In one embodiment, the anti-cancer agent can be mixed with additionalsubstances to enhance their effectiveness.

Methods of Administration

In an alternative embodiment, the disclosed compounds and the disclosedpharmaceutical compositions are administered by a particular method asan anti-cancer, or anti-inflammatory. Such methods include, amongothers, (a) administration though oral pathways, which administrationincludes administration in capsule, tablet, granule, spray, syrup, orother such forms; (b) administration through non-oral pathways, whichadministration includes administration as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like; administration via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or thelike; as well as (c) administration topically, (d) administrationrectally, or (e) administration vaginally, as deemed appropriate bythose of skill in the art for bringing the compound of the presentembodiment into contact with living tissue; and (f) administration viacontrolled released formulations, depot formulations, and infusion pumpdelivery. As further examples of such modes of administration and asfurther disclosure of modes of administration, disclosed herein arevarious methods for administration of the disclosed compounds andpharmaceutical compositions including modes of administration throughintraocular, intranasal, and intraauricular pathways.

The pharmaceutically effective amount of the compositions that includethe described compounds required as a dose will depend on the route ofadministration, the type of animal, including human, being treated, andthe physical characteristics of the specific animal under consideration.The dose can be tailored to achieve a desired effect, but will depend onsuch factors as weight, diet, concurrent medication and other factorswhich those skilled in the medical arts will recognize. In a typicalembodiment, a compound represented by Formulae I and II can beadministered to a patient in need of an anti-cancer agent, until theneed is effectively reduced or preferably removed.

In practicing the methods of the embodiment, the products orcompositions can be used alone or in combination with one another, or incombination with other therapeutic or diagnostic agents. These productscan be utilized in vivo, ordinarily in a mammal, preferably in a human,or in vitro. In employing them in vivo, the products or compositions canbe administered to the mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, vaginally, nasally or intraperitoneally,employing a variety of dosage forms. Such methods may also be applied totesting chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired affects and thetherapeutic indication. Typically, dosages can be between about 10 mg/kgand 100 mg/kg body weight, preferably between about 100 mg/kg and 10mg/kg body weight. Alternatively dosages can be based and calculatedupon the surface area of the patient, as understood by those of skill inthe art. Administration is preferably oral on a daily or twice dailybasis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975, which is incorporated herein by reference in its entirety. Itshould be noted that the attending physician would know how to and whento terminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above can be used in veterinary medicine.

Depending on the specific conditions being treated, such agents can beformulated and administered systemically or locally. A variety oftechniques for formulation and administration can be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990), which is incorporated herein by reference in itsentirety. Suitable administration routes may include oral, rectal,transdermal, vaginal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

For injection, the agents of the embodiment can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theembodiment into dosages suitable for systemic administration is withinthe scope of the embodiment. With proper choice of carrier and suitablemanufacturing practice, the compositions disclosed herein, inparticular, those formulated as solutions, can be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the embodiment to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly can be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents can be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules canbe directly administered intracellularly.

Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration can be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions can be manufactured in amanner that is itself known, for example, by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, can be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, dogs or monkeys, can be determined using known methods. Theefficacy of a particular compound can be established using several artrecognized methods, such as in vitro methods, animal models, or humanclinical trials. Art-recognized in vitro models exist for nearly everyclass of condition, including the conditions abated by the compoundsdisclosed herein, including cancer, cardiovascular disease, and variousimmune dysfunction, and infectious diseases. Similarly, acceptableanimal models can be used to establish efficacy of chemicals to treatsuch conditions. When selecting a model to determine efficacy, theskilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, and regime. Ofcourse, human clinical trials can also be used to determine the efficacyof a compound in humans.

As will be understood by one of skill in the art, “need” is not anabsolute term and merely implies that the patient can benefit from thetreatment of the anti-cancer agent in use. By “patient” what is meant isan organism that can benefit by the use of an anti-cancer agent.

“Therapeutically effective amount,” “pharmaceutically effective amount,”or similar term, means that amount of drug or pharmaceutical agent thatwill result in a biological or medical response of a cell, tissue,system, animal, or human that is being sought. In a preferredembodiment, the medical response is one sought by a researcher,veterinarian, medical doctor, or other clinician.

In one embodiment, a described compound, preferably a compound havingany one of Formulas I and II, including those as described herein, isconsidered an effective anti-cancer agent if the compound can influence10% of the cancer cells, for example. In a more preferred embodiment,the compound is effective if it can influence 10 to 50% of the cancercells. In an even more preferred embodiment, the compound is effectiveif it can influence 50-80% of the cancer cells. In an even morepreferred embodiment, the compound is effective if it can influence80-95% of the cancer cells. In an even more preferred embodiment, thecompound is effective if it can influence 95-99% of the cancer cells.“Influence” is defined by the mechanism of action for each compound.

EXAMPLES General Synthetic Procedures General Procedure A—HATU MediatedAmide Bond Formation

To an acid (1.1 eq with respect to amine) in anhydrous DMF was addedHATU (1 eq with respect to acid) and DIEA (2 eq with respect to acid)and the mixture was stirred at room temperature for 1 minute. Themixture was then added to a solution of amine in DMF and the reactionmixture was stirred at room temperature till the completion of thereaction (monitored by LC/MS). The solvent was removed under reducedpressure and the residue was optionally purified by reverse phase HPLCto give final pure product.

General Procedure B—DIC/HOAt Mediated Amide Bond Formation

To a stirred solution of carboxylic acid (1.1 eq), amine and HOAt (1.1eq) in anhydrous DMF was added DIC (1.1 eq) and the reaction mixture wasstirred at room temperature. Upon completion (monitored by LC/MS), thesolvent was removed under reduced pressure and the residue wasoptionally purified by reverse phase HPLC to give final pure product.

General Procedure C—Removal of Acid Sensitive Protecting Groups (Boc,THP, t-Bu) Using HCl/Dioxane

The acid sensitive protecting groups containing compound was dissolvedin 4N HCl/dioxane and the mixture was stirred at room temperature for 2h. The solution was then concentrated under reduced pressure and theresidue was washed twice with cold ether. Purification was carried outon reverse phase HPLC if necessary.

General Procedure D—Removal of Fmoc Group

The Fmoc containing compound was dissolved in 2-5% piperidine in DMF.The mixture was stirred at room temperature for 1 h. The solvents wereremoved under reduced pressure. Purification was carried out on reversephase HPLC if necessary.

General Procedure E—Reductive Alkylation

An amine was dissolved in DMF and aldehyde (5 eq) was added, followed byaddition of sodium cyanoborohydride (5 eq). HOAc was added to adjust thepH of the reaction mixture to 4-5. The mixture was stirred at roomtemperature till completion (1-4 h, monitored by HPLC). Purification wascarried out on reverse phase HPLC if necessary.

General Procedure F—Saponification—Removal of Me/Ft from Esters

To a stirred solution of an ester in MeOH was added 1M aq. solution ofLiOH till pH of the mixture was about 13-14 and the reaction mixture wasstirred at room temperature till completion (˜16 h, monitored by HPLC).Citric acid (˜10% aq,) was added to neutralize the reaction and thesolvents were removed under reduced pressure. The crude product wasoptionally purified by RP-HPLC or used directly in the next step.

General Procedure G—Activation of a Hydroxyl/Phenol Group withBis(p-Nitrophenyl)Carbonate

To a stirred solution of an alcohol/phenol in THF/DMF (2/1) was addedbis(p-nitrophenyl) carbonate (3-5 eq), followed by DIEA (2-4 eq) and thereaction mixture was stirred at room temperature until most of thestarting material was consumed. The progress of the reaction wasmonitored by LC/MS. The crude product was optionally purified by flashcolumn chromatography or by precipitation and washing.

General Procedure H—Reaction of an Amine with a Cyclic Anhydride(Glutaric Anhydride or Succinic Anhydride)

An amine containing compound was dissolved in DMF. Glutaric anhydride (3eq) was added, followed by addition of DIEA (4 eq). The reaction mixturewas stirred at room temperature until most of the starting material wasconsumed. The progress of the reaction was monitored by LC/MS. The crudeproduct was purified by RP-HPLC to yield the pure carboxylic acid.

General Procedure I—Formation of Carbamate with p-Nitrophenyl Carbonate(e.g. FmocVC-PAB-PNP)

An amine containing compound was dissolved in DMF and alkyl/arylp-nitrophenyl carbonate (1.5 eq) was added, followed by addition of DIEA(2 eq) and HOBt (cat., 5%). The reaction mixture was stirred at roomtemperature until most of the amine was consumed. The progress of thereaction was monitored by LC/MS. The crude product was optionallypurified by RP-HPLC to yield the pure carbamate.

General Procedure I—Formation of an Activated Ester (e.g. NHS) from anAcid

An acid was dissolved in DCM and DMF was added to aid dissolution ifnecessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl(1.5 eq). The reaction mixture was stirred at room temperature for 1 huntil most of the acid was consumed. The progress of the reaction wasmonitored by RP-HPLC. The mixture was then diluted with DCM and washedsuccessively with citric acid (aq. 10%) and brine. The organic layer wasdried and concentrated to dryness. The crude product was optionallypurified by RP-HPLC or silica gel column chromatography.

General Scheme for Active Agent Conjugates Formation Conjugation MethodA. Conjugation on Lys Residues Via an Activated Carboxylic Acid

Conjugation Method B. Conjugation on Lys Residues Via ReductiveAlkylation with an Dialdehyde

Conjugation Method C. Conjugation on Individual Cys Side Chain EmployingMaleimide Chemistry

Conjugation Method D. Conjugation on Two Cys Side Chains by Forming aCyclic Structure

Conjugation Method E. Conjugation on Carbonyl (Ketone/Aldehyde) BearingBiologics by Formation of an Oxime Moiety

Conjugation Method F. Conjugation on Azide Bearing Biologics UsingCopper-Free Click Chemistry:

Experimental Description Step 1. Drug-Linker Construct Synthesis (-L²-D)Methods of Drug-Linker Construct Synthesis, but not Limited to:

Method 1-1: Linker and Drug Connected Via a Carbamate Bond. TheFollowing General Procedures were Employed:

General Procedure G and I for Activation and Carbamate Formation GeneralProcedure C, D, and F for Removal of Protective Groups for FurtherDerivatization.

Method 1-2: Linker and Drug Connected Via Reductive Alkylation (GeneralProcedure E)

Method 1-3. Active Molecule Containing a Carboxylic Acid MoietyConnected to an Alkoxyamino Linker Via Formation of Hydroxamate (GeneralProcedure A or B), Followed by Removal of Protective Groups.

For active molecules that are hydroxamic acids, the above method stillcan be employed since the construct will release hydroxamic acid underenzymatic cleavage conditions. The reaction needs to start from itscorresponding carboxylic acid.

Step 2. Introducing Functional Groups to L1-(L2-D)

Methods to Introduce Functional Groups that Suitable for ConjugationReaction, but No Limited to:Method 2-1. Compounds Bearing Free Amino Group to React with an CyclicAnhydride to Introduce Carboxylic Acid (General Procedure H).

Method 2-2. Compounds Bearing Free Amino Group to React with a Di-Acidto Introduce Carboxylic Acid (General Procedure B).

Method 2-3. Removal of Carboxylic Acid Protective Group to Reveal theFree Carboxylic Acid (General Procedure C, F)

Method 2-4. Reductive Alkylation of a Primary Amine with a DialdehydeBearing a Carboxylic Acid Moiety (General Procedure E)

The amine (NH₂-Ahx-Maytansinol) (20 mg) was dissolved in acetonitrile (2mL) and 1 mL of NaOAc buffer (100 mM, pH=4.0) was added. The dialdehyde(0.5 M solution in water, 0.2 mL) was added, followed by NaCNBH₃ (10mg). The reaction mixture was stirred at room temperature for 30 minsand purified directly by RP-HPLC to give the desired acid (16 mg) as awhite solid after lyophilization. MS found: 790.5 (M+H)⁺.

The dialdehyde carboxylic acid was synthesized according to thefollowing scheme:

The ester, 2H-Pyran-4-carboxylic acid, 2-ethoxyl-3,4-dihydro-ethyl esterwas synthesized according to a literature procedure (ChemCommunications, (1) 25-26, 1998) was saponified using General procedureF, followed by treatment with 1N aq. HCl at room temperature for 1 h.The aq. solution of dialdehyde was used directly without furtherpurification.

Method 2-5. Introduction of o-Phenylenediamine Moiety

The 3-nitro-4-amino benzoic acid was incorporated using standardamidation reaction (General procedure B). The nitro group was reducedusing sodium dithionite (3 eq) in acetonitrile/water to give the desiredo-phenylene diamine. MS found: 1504.8 (M+H)⁺.

Step 3. Introducing the Final Functional Groups Prior to ConjugationMethods of Introduction of Final Reactive Group Prior to ConjugationReaction, but not Limited to: Method 3-1. Activation of a CarboxylicAcid to its Corresponding Activated Form

G is a leaving group selected from —F, —Cl, —Br, —I, —N₃, —OR (R=alkyl,aryl, heteroaryl, substituted aryl, substituted heteroaryl), SR(R=alkyl, aryl heteroaryl, substituted aryl, substituted heteroaryl),—ON(R¹)R², (R¹, R² are each independently selected from —(C═O)—R, R=H,alkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl or R¹and R² are connected to form a cyclic structure, or R¹=R²=(═C—R),RC(═O)O—, and RSO₂—O— (R=alkyl, aryl, heteroaryl, substituted aryl,substituted heteroaryl).

The carboxylic acid can be activated using a variety of methods toafford an activated form. For example, the carboxylic acid can beactivated using the following methods: A) Tetrahedron 61 (2005)10827-10852; B) Beckwith, A. L. J. In The Chemistry of Amides; Zabicky,J., Ed.; Synthesis of Amides; Interscience: London, 1970; pp 105-109; C)Handbook of Reagents for Organic Synthesis: Activating Agents andProtecting Groups; Pearson, A. J., Roush, W. R., Eds.; Wiley: New York,1999; pp 370-373; D) Lloyd-Williams, P., Albericio, F., and Giralt, E.(1997). Chemical approaches to the synthesis of peptide and proteins(Series ed. C. W. Rees). CRC Press, New York; E) Peptide chemistry: Apractical textbook: By M Bodansky. Springer-Verlag, Heidelberg. 1988;and F) The practice of peptide synthesis, 2nd ed., by M. Bodansky and A.Bodansky, Springer-Verlag, New York, each of which is incorporatedherein by reference in its entirety.

Method 3-2. Introducing a maleimido moiety

a. Via Amidation (General Procedure A, or B, or from an Activated EsterBearing a Maleimido Moiety)

b. Converting an Existing Amino Group to Maleimide Directly UsingN-Methoxy-Carbonylmaleimide

The amine (0.1 mmol) was dissolved in acetonitrile/water (6/4, v/v, 3mL). The mixture was cooled in an ice-water bath and treated with sat.aq. NaHCO₃ (0.5 mL), followed by N-methoxycarbonylmaleimide (0.12 mmol).The mixture was stirred at room temperature for 1 h. The pH was adjustedto 6-7 with citric acid and the solution was concentrated. The residuewas purified by RP-HPLC to yield the desired maleimide as a white powderafter lyophilization (58%). MS found: 1091.2 (M+H)⁺.

Method 3-3. Formation of Dibromomethyl Quinoxaline

The o-phenylenediamino compound (12 mg) was dissolved inacetonitrile/water. Dibromomethyl diketone (10 mg) was added. Themixture was stirred at room temperature for 10 min and purified directlyby RP-HPLC to give the desired quinoxaline as a white powder (12 mg)after lyophilization. MS found 1713.0 (M+H)⁺.

Method 3-4. Incorporation of Hydroxylamine Moiety

Method 3-5. Introduction of Cyclooctyne Moiety

Example I. Synthesis of Compound 10

To a solution of compound 1 (23.4 g, 81.53 mmol) in dry EtOH (200 mL)was added SOCl₂ (100 mL) at 0° C. The mixture was stirred for overnightand the solvent was removed by evaporation in vacuo. The residue wasimmediately used for the next step without further purification. To asolution of compound 2 (81.53 mmol), compound 3 (50 g, 163.1 mmol) indry DMF (150 mL) was added DEPC (15.9 g, 97.8 mmol), TEA (41 g, 0.408mol) at 0° C. The mixture was stirred for 2 h at 0° C. Then the mixturewas stirred overnight at room temperature. Solvent was removed byevaporation in vacuo. The residue was diluted with ethyl acetate-toluene(2:1, 900 ml) and washed with 1M KHSO₄, water, sat. NaHCO₃, and brine.The organic layer was dried and concentrated to give a residue, whichwas purified by column (hexanes:ethylacetate:DCM=5:1:1) to give 38 g ofcompound 4.

To a solution of Boc-Val-OH (30.6 g, 0.142 mol), compound 5 (from 25 gof compound 4) in DCM (400 mL) was added BrOP (28 g, 70.87 mmol), DIEA(30 g, 0.236 mol) at 0° C. The mixture was shielded from light andstirred for 0.5 h at 0° C. Then the mixture was stirred for 48 h at roomtemperature. The solvent was removed by evaporation in vacuo. Theresidue was diluted with ethyl acetate-toluene (3:1, 900 mL) and washedwith 1M KHSO₄, water, sat. NaHCO₃, and brine. The organic layer wasdried and concentrated to give a residue, which was purified by silicagel column (hexanes:ethylacetate:DCM=3:1:1) to give 22 g of compound 7.

To a solution of compound 7 (40 g, 66.7 mmol) in THF (600 mL) was addeda mixture of LiOH (14 g, 0.33 mol) in water (300 mL) below 10° C. Themixture was stirred for 5 days at 25° C. THF was removed by evaporation.The aqueous layer was washed with Et₂O (200 mL×3). The aqueous layer wasacidified to pH 2 with 1N HCl at 0° C., the mixture was extracted withethyl acetate and the organic layer was washed with water and brine. Theorganic layer was dried and concentrated to give a residue, which waspurified by Prep-HPLC to give 14 g of compound 8.

To a solution of compound 8 (3 g) in DCM (100 mL) was added compound 9(3 g, prepared according to General procedure J from Boc-N-Me-Val-OHusing EDC and pentafluorophenol). DIEA (2.6 mL) was added, followed byHOBt (cat. 100 mg) and the reaction mixture was stirred at roomtemperature for 16 h. The solvents were removed under reduced pressureand the residue was purified on a silica gel column to give compound 10as a white powder (3.1 g). MS m/z Calcd for C₃₅H₆₄N₄O₉ 684.5, Found707.6 ([M+Na]⁺).

Example II. Preparation of Cytotoxic Compounds—Sulfonamide Derivatives

-L1-(L2-D) MS Synthetic Conj. found ID Cytotoxic compound (D) methodMethod [M + H]⁺ 13

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F835.6 14

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F809.6 16

1-1, 1-2 2-1, 2-2 2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B, C, D, E, F 801.718

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F836.6 30

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F827.5 31

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F807.8 32

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F793.4 35

1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D,E, F 839.6 37

1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D,E, F 865.7 121

1-1, 1-2 2-1, 2-2 2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B, C, D, E, F 838.3122

1-1, 1-2 2-1, 2-2 2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B, C, D, E, F 864.544

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 1011.8 45

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 1037.5 123

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F823.2 124

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F837.5 125

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F835.7 126

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F871.7 127

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F851.3 128

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F817.5 129

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F817.1 130

1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F818.4

Example IIa. Synthesis of Compound 13, 14, 16, 18

The amino acid sulfonamide derivatives 11, 15, 17, 19 were synthesizedaccording to previously reported procedure (ARKIVOC 2004 (xii) 14-22)using Boc protected amino acid and cyclopropyl/methyl sulfonamide,followed by removal of Boc (General procedure C)

Example IIa-1. Synthesis of Compound 13

Compound 13 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 1) and amine II1,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 13 as a white powderafter lyophilization. MS m/z Calcd for C₄₂H₇₀N₆O₉S 834.5, Found 835.6([M+H]⁺).

Example IIa-2. Synthesis of Compound 14

Compound 14 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 15,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 14 as a white powderafter lyophilization. MS n/z Calcd for C₄₀H₆₈N₆O₉S 808.5, Found 809.6([M+H]⁺).

Example IIa-3. Synthesis of Compound 16

Compound 16 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 17,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 16 as a white powderafter lyophilization. MS nm/z Calcd for C₃₉H₇₂N₆O₉S 800.5, Found 801.7([M+H]⁺).

Example IIa-4. Synthesis of Compound 18

Compound 18 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 19,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 18 as a white powderafter lyophilization. MS m/z Calcd for C₄₁H₆₉N₇O₉S 835.5, Found 836.6([M+H]⁺).

Example IIb. Synthesis of Compound 27

To a stirred solution of compound 1 (2.9 g, 10 mmol) and compound 11(HCl salt, 3 g) in anhydrous DMF (100 mL) was added HOBt (1.54 g) andEDC.HCl (2 g). DIEA (20 mmol) was added and the mixture was stirred atroom temperature for 16 h. The solvents were removed under reducedpressure and the residue was dissolved in ethyl acetate (500 mL) andwashed successively with Citric acid (10%, aq., 200 mL). NaHCO3 (sat.aq., 200 mL) and brine. The organic layer wad dried and evaporated todryness to give compound 20 as a syrup which was treated with 6N HCl iniPr-OH (100 mL) for 1 h to give compound 21 after concentration.Compound 21 was used directly without further purification.

To a solution of Boc-Val-OH (2.5 g), compound 22 in DCM (150 mL) wasadded PyBrOP (11 mmol), DIEA (22) at 0° C. The mixture was shielded fromlight and stirred for 0.5 h at 0° C. Then the mixture was stirred for 24h at room temperature. The solvent was removed by evaporation in vacuo.The residue was diluted with ethyl acetate (300 mL) and washed with 1MKHSO₄, water, sat. NaHCO₃, and brine. The organic layer was dried andconcentrated to give a residue, which was purified by silica gel column(hexanes: ethyl acetate) to give 3.2 g of compound 23.

The compound 23 (3 g) was dissolved in DCM (100 mL) and TFA (30 mL) wasadded. The mixture was stirred at room temperature for 2 h andconcentrated under reduced pressure. The residue was dissolved indioxane (200 mL). Sat. aq. NaHCO₃ (80 mL) was added, followed by Bocanhydride (2.2 g). The mixture was stirred at room temperature for 4 hand neutralized to pH 3-4 using 1N HCl. Solvents were removed and theresidue was dissolved in ethyl acetate, which was washed with water andbrine. The organic layer was concentrated and the residue was purifiedon a silica gel column to give compound 25 as a syrup.

Compound 27 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between compound 21 and amine 25, followed by removal of Boc (Generalprocedure C). The final compound was purified by reverse phase HPLC togive compound 27 as a white powder after lyophilization. MS m/z Calcd.for C₃₆H₅₉N₅O₈S 721.4, Found 722.5 ([M+H]⁺).

Example IIc. Synthesis of Compounds 30, 31, and 31

Example IIc-1. Synthesis of Compound 30

Compound 30 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between β-Cl—N-Boc-Ala-OH (compound 28) and amine 27, followed byremoval of Boc (General procedure C). The final compound was purified byreverse phase HPLC to give compound 30 as a white powder afterlyophilization. MS m/z Calcd for C₃₉H₆₃ClN₆O₉S 826.4, Found 827.5([M+H]⁺).

Example IIc-2. Synthesis of Compound 31

Compound 31 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between N-Boc-Aib-OH and amine 27 followed by removal of Boc (Generalprocedure C). The final compound was purified by reverse phase HPLC togive compound 31 as a white powder after lyophilization. MS m/z Calcd.for C₄₀H₆₆N₆O₉S 806.5, Found 807.8 ([M+H]⁺).

Example IIc-3. Synthesis of Compound 32

Compound 32 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between N-Boc-Sar-OH and amine 27, followed by removal of Boc (Generalprocedure C). The final compound was purified by reverse phase HPLC togive compound 32 as a white powder after lyophilization. MS nm/z Calcd.for C₃₉H₆₄N₆O₉S 792.5, Found 793.4 ([M+H]⁺).

Example IId. Synthesis of Compounds 35 and 37

The amino acid sulfonamide derivatives 33 and 36 were synthesizedaccording to previously reported procedure (WO 2007146695) using Bocprotected amino acid and cyclopropyl/methyl sulfonamide, followed byremoval of Boc (General procedure C)

Example IId-1. Synthesis of Compound 35

Compound 35 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 33,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 35 as a white powderafter lyophilization. MS m/z Calcd for C₄₁H₇₀N₆O₁₀S 838.5, Found 839.6([M+H]⁻).

Example IId-2. Synthesis of Compound 37

Compound 37 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 36,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 37 as a white powderafter lyophilization. MS m/z Calcd for C₄₃H₇₂N₆O₁₀S 864.5, Found 865.7([M+H]⁺).

Example IIe. Synthesis of Compounds 44 and 45

Example IIe-1. Synthesis of Compound 44

The phenol 35 (1 mmol) was treated with 3 eq ofbis(p-nitrophenyl)carbonate to form the activated carbonate 39 (generalprocedure G). The crude product was used directly without furtherpurification. 6-Aminohexanoic acid (5 eq) was dissolved in sat. aq.NaHCO₃ (5 mL) and the solution was added. The reaction mixture wasstirred at room temperature for 16 h. Citric acid (aq. 10%) was added toacidify the reaction (pH=4-5) and then diluted with EtOAc (150 mL).Organic layer was dried (over Na₂SO₄) and concentrated to give the crudeproduct 40 which underwent the following procedures: removal of Boc(General procedure C), reductive alkylation using HCHO (Generalprocedure E), DIC/HOAt mediated amide bond formation (General procedureB) between compound 42 and THP—O—NH₂, followed by removal of THP(General procedure C, using 4N aq. HCl). The final compound was purifiedby reverse phase HPLC to give compound 44 as a white powder afterlyophilization. MS m/z Calcd for C₄H₈₂N₈₀O₃S 1010.6, Found 1011.8([M+H]⁺).

Example IIe-2. Synthesis of Compound 45

The compound 45 was synthesized according to the same procedures asdescribed for the synthesis of compound 44. The final compound waspurified by reverse phase HPLC to give compound 45 as a white powderafter lyophilization. MS m/z Calcd for C₅₀H₈₄N₈O₁₃S 1036.6, Found 1037.5([M+H]⁺).

Example III Synthesis of Cytotoxic Compounds

-L¹-(L²-D) MS Synthetic Conjugation found ID Cytotoxic compound (D)method Method [M + H]⁺ 49

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 712.5 50

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 738.5 52

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 784.7 53

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 788.7 54

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 772.5 137

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 682.4 138

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 698.6

Example III-1. Synthesis of Compound 49

Compound 49 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and NH₂-Leu-OMe,followed by removal of Boc (General procedure C), reductive alkylationwith HCHO (General procedure E) and saponification to remove methylgroup from ester (General procedure F). The final compound was purifiedby reverse phase HPLC to give compound 49 as a white powder afterlyophilization. MS m/z Calcd for C₃₇H₆₉N₅O₈ 711.5, Found 712.5 ([M+H]⁺).

Example III-2. Synthesis of Compound 50

Compound 50 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 51, followedby removal of Boc (General procedure C), reductive alkylation with HCHO(General procedure E) and saponification to remove methyl group fromester (General procedure F). The final compound was purified by reversephase HPLC to give compound 51 as a white powder after lyophilization.MS m/z Calcd for C₃₉H₇₁N₅O₈ 737.5, Found 738.5 ([M+H]⁺).

Compound 51 was synthesized according to literature procedures (J. Org.Chem., 2001, 66, 7355-7364)

Example III-3. Synthesis of Compound 52

Compound 52 was synthesized using the general procedures described aboveas following: HATU mediated amide bond formation (General procedure A)between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 1) and amine 22, followedby removal of Boc and t-Bu (General procedure C), reductive alkylationwith HCHO (General procedure E). The final compound was purified byreverse phase HPLC to give compound 52 as a white powder afterlyophilization. MS m/z Calcd for C₄₁H₆₆N₅O₈ 783.6, Found 784.7 ([M+H]⁺).

Example III-4. Other Compounds Synthesized Using the Same Procedures asDescribed for the Synthesis of Compound 49

For compound 53: MS m/z Calcd for C₄₃H₇₃N₅O₈ 787.6, Found 788.7([M+H]⁺).

For compound 54: MS m/z Calcd for C₄₂H₆₉N₅O₈ 771.5 Found 772.5 ([M+H]⁺).

Example III-5

Example III-5a. Synthesis of Compound 137

The intermediate 136 was synthesized using the general proceduresdescribed above as following (0.1 mmol scale): HATU mediated amide bondformation (General procedure A) between Boc-N-Mc-Val-Val-Dil-Dap-OH(compound 1) and amine 134, followed by removal of Boc (Generalprocedure C). The HCl salt 11 was dissolved in water (5 mL) and hexane(5 mL) was added. Phenylboronic acid (10 eq) was added and thesuspension was stirred vigorously at room temperature for 1 h. Hexanelayer was removed and fresh hexane (5 mL) was added. The mixture wasagitated for another 2 h and the aqueous layer was collect andconcentrated. The final compound was purified by reverse phase HPLC togive compound 12 as a white powder after lyophilization. MS m/z Calcdfor C₃₄H₆₄BN₅O₈ 681.5, Found 682.4 ([M+H]⁺).

Example III-5b. Synthesis of Compound 138

The compound 138 was synthesized employing the same sequence asdescribed for the preparation of compound 137. (compound 139 as astarting material). It was obtained as a white powder after RP-HPLCpurification and lyophilization. MS m/z Calcd for C₃₅H₆₈BN₅O₈ 697.5,Found 698.6 ([M+H]⁺).

Example IV. Preparation of Cytotoxic Compounds—Hydroxamic AcidDerivatives

-L¹-(L²-D) MS Synthetic Conjugation found ID Cytotoxic compound (D)method Method [M + H]⁺ 56

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 727.6 57

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 761.6 59

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 489.5 58

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 743.5 60

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 753.5 61

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 799.5 73

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 719.3 74

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 759.4 75

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 725.2 76

1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 685.7

Example IV-1. Synthesis of Compound 56

Compound 56 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Me₂-Val-Val-Dil-Dap-LeuOH (compound 49) and THP—O—NH₂,followed by removal of THP (General procedure C, using 4N aq. HCl). Thefinal compound was purified by reverse phase HPLC to give compound 56 asa white powder after lyophilization. MS m/z Calcd for C₃₇H₇₀N₆O₈ 726.5,Found 727.6 ([M+H]⁺).

Example IV-2. Synthesis of Compound 57

Dimethyl Auristatin F was synthesized from compound 10 and NH₂-Phe-OMeusing the synthetic procedures described above for the synthesis ofcompound 49.

Compound 57 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between dimethyl auristatin F and THP—O—NH₂, followed by removal ofTHP (General procedure C, using 4N aq. HCl). The final compound waspurified by reverse phase HPLC to give compound 57 as a white powderafter lyophilization. MS n/z Calcd for C₄₀H₆₈N₆O₈ 760.5, Found 761.6([M+H]⁺).

Example IV-3. Synthesis of Compound 58

Compound 58 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Tubulysin (J. Am. Chem. Soc., 2006, 128 (50), pp 16018-16019)and THP—O—NH₂, followed by removal of THP (General procedure C, using 4Naq. HCl). The final compound was purified by reverse phase HPLC to givecompound 45 as a white powder after lyophilization. MS m/z Calcd forC₃₈H₅₈N₆O₇S 742.4, Found 743.5 ([M+H]⁺).

Example IV-4. Synthesis of Compound 59

Compound 59 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between HTI-286 (Bioorg Med Chem Lett. 2004, 14(16):4329-32) andTHP—O—NH₂, followed by removal of THP (General procedure C, using 4N aq.HCl). The final compound was purified by reverse phase HPLC to givecompound 59 as a white powder after lyophilization. MS m/z Calcd forC₂₇H₄₄N₄O₄ 488.3, Found 489.5 ([M+H]⁺).

Example IV-5. Synthesis of Compound 60

Compound 60 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 50 and THP—O—NH₂, followed by removal of THP(General procedure C, using 4N aq. HCl). The final compound was purifiedby reverse phase HPLC to give compound 47 as a white powder afterlyophilization. MS m/z Calcd for C₃₉H₇₂N₆O₈ 752.5, Found 753.5 ([M+H]⁺).

Example IV-6. Synthesis of Compound 61

Compound 61 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 52 and THP—O—NH₂, followed by removal of THP(General procedure C, using 4N aq. HCl). The final compound was purifiedby reverse phase HPLC to give compound 61 as a white powder afterlyophilization. MS m/z Calcd for C₄₁H₇₈N₆O₉ 798.5, Found 799.5 ([M+H]⁺).

Example V. Synthesis of Alkoxyamine Linkers 65, 66, 67, and 68

ID Structure 65

66

67

68

Example V-1. Synthesis of Compound 65

To a stirred solution of Fmoc-VA-PAB (62) (Bioconjugate Chem., 2002, 13,855-859) (9 g, 15 mmol) in THF (200 mL) was added thionyl chloride (18mmol) dropwise. After the addition was complete, the reaction mixturewas stirred at room temperature for 1 h. TLC analysis (ethylacetate/hexane, 1/1, v/v) showed the completion of the reaction. Thesolvents were removed under reduced pressure and the residue was washedwith hexanes (100 mL) to give compound 63 as a slightly yellowish solid(8.8 g).

Compound 63 (6.2 g, 10 mmol) was dissolved in anhydrous DMF (100 mL).N-Hydroxy-phthalimide (3.2 g, 20 mmol) was added, followed by solidNaHCO₃ (3.4 g, 40 mmol). The reaction mixture was stirred at roomtemperature for 48 h. TLC analysis showed that most of compound 61 wasconsumed. The reaction was then diluted with ethyl acetate (500 mL) andwashed successively with sat. aq. NaHCO₃ (3×200 mL) and brine (200 mL).The organic layer was dried and concentrated to give compound 64 as atan solid, which was used directly without further purification.

The crude compound 64 from previous step was dissolved in DMF (100 mL).HOAc (6 mL) was added, followed by hydrazine hydrate (5 mL). Thereaction was stirred at room temperature for 1 h. LC/MS showed thecompletion of the reaction. The reaction mixture was then poured into abeaker containing IL of water under stirring. The precipitated solid wascollected via filtration and washed twice with water to give compound 65as a white solid (purity >85%, can be used directly). Pure compound 63was obtained after RP-HPLC purification. MS m/z Calcd for C₃₀H₃₄N₄O₅530.3, Found 531.4 ([M+H]⁺).

Example V-2. Synthesis of Compound 66

Compound 66 was synthesized starting from compound Fmoc-VC-PAB(Bioconjugate Chem., 2002, 13, 855-859) using the procedures describedabove for the synthesis of compound 63. MS m/z Calcd for C₃₃H₄₀N₆O₆616.3, Found 617.5 ([M+H]⁺).

Example V-3. Synthesis of Compound 67

Compound 64 was synthesized starting from compound Fmoc-A-PAB(synthesized according to the procedure reported: Bioconjugate Chem.,2002, 13, 855-859) using the procedures described above for thesynthesis of compound 63. MS m/z Calcd for C₂₅H₂₅N₃O₄ 431.2, Found 432.6([M+H]⁺).

Example V-4. Synthesis of Compound 68

Compound 68 was synthesized starting from compound Fmoc-Ahx-PAB usingthe procedures described above for the synthesis of compound 68. MS m/zCalcd for C₂₈H₃₁N₃O₄ 473.2, Found 474.3 ([M+H]⁺).

Example VIII. Synthesis of -L¹-(L²-D)-

MS Conj. found ID Structure of -L¹-(L²-D) Method [M + H]⁺ 83

A 1150.9 84

A 1237.1 85

A 1051.9 86

A 1093.9 88

A 1091.9 89

A 1118.0 90

A 1263.1 91

A 1274.8 93

A 1278.9 92

A 1218.9 94

A 878.4

Example VIII-1. Synthesis of Compound 83

Compound 83 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Auristatin F and compound 65, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 83 as a white powder after lyophilization. MS m/z Calcdfor C₆₀H₉₅N₉O₁₃ 1149.7, Found 1150.9 ([M+H]⁺).

Example VIII-2 Synthesis of Compound 84

Compound 84 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Auristatin F and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 84 as a white powder after lyophilization. MS m/z Calcdfor C₆H₁₀₁N₁₁O₁₄ 1235.8, Found 1237.1 ([M+H]⁺).

Example VIII-3. Synthesis of Compound 85

Compound 85 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Auristatin F and compound 67, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 85 as a white powder after lyophilization. MS n/z Calcdfor C₅₅H₈₆N₈O₁₂ 1050.6, Found 1051.9 ([M+H]⁺).

Example VIII-4. Synthesis of Compound 86

Compound 86 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Auristatin F and compound 68, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 86 as a white powder after lyophilization. MS m/z Calcdfor C₅₈H₉₂N₈O₁₂ 1092.7, Found 1093.9 ([M+H]⁺).

Example VIII-5. Synthesis of Compound 88

Compound 88 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 49 and compound 67, followed by removal of Fmoc(General procedure D), and amide formation with acid 87 using HATU(General procedure A, 3 eq of acid 87 and 1 eq of HATU were used). Thefinal compound was purified by reverse phase HPLC to give compound 88 asa white powder after lyophilization. MS m/z Calcd for C₅₅H₉₄N₅O₁₄1090.7, Found 1091.9 ([M+H]⁺).

Example VIII-6. Synthesis of Compound 89

Compound 89 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 50 and compound 67, followed by removal of Fmoc(General procedure D), and amide formation with acid 87 using HATU(General procedure A, 3 eq of acid 87 and 1 eq of HATU were used). Thefinal compound was purified by reverse phase HPLC to give compound 89 asa white powder after lyophilization. MS m/z Calcd for C59H96N8O141116.7, Found 1118.0 ([M+H]⁺).

Example VIII-7. Synthesis of Compound 90

Compound 90 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 54 and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 90 as a white powder after lyophilization. MS m/z Calcdfor C₆₅H₁₀N₁₁O₁₄ 1261.8, Found 1263.1 ([M+H]⁺).

Example VIII-8. Synthesis of Compound 91

Compound 91 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 52 and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 91 as a white powder after lyophilization. MS m/z Calcdfor C₆₄H₁₁₁N₁₁O₁₅ 1273.8, Found 1274.8 ([M+H]f.

Example VIII-9. Synthesis of Compound 92

Compound 92 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Tubulysin M and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 92 as a white powder after lyophilization. MS m/z Calcdfor C₆₁H₉₁N₁₁O₁₃S 1217.7, Found 1218.9 ([M+H]⁺).

Example VIII-10. Synthesis of Compound 93

Compound 93 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound 53 and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 93 as a white powder after lyophilization. MS m/z Calcdfor C₆₆H₁₀₇N₁₁O₁₄ 1277.8, Found 1278.9 ([M+H]⁺).

Example VIII-11. Synthesis of Compound 94

Compound 94 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between compound HTI-286 and compound 66, followed by removal of Fmoc(General procedure D), and reaction with glutaric anhydride (Generalprocedure H). The final compound was purified by reverse phase HPLC togive compound 94 as a white powder after lyophilization. MS min Calcdfor C₄₇H₇₁N₇₁O₉ 877.5, Found 878.4 ([M+H]⁺).

Example IX Synthesis of -L1-(L2-D)-

MS found Conj. [M + ID Structure of -L¹-(L²-D) Method H]⁺ 97

A 1354.9 98

A 1243.8 99

A 995.8 102

A 1135.6 103

A 1209.8 104

A 1486.9 105

A 1512.9 131

A 1444.0 110

A 1020.8 113

A 923.7 114

A 1034.7 115

A 1325.9 132

A 976.3

Example IX-1. Synthesis of Compound 97

Compound 97 was synthesized using the general procedures described aboveas following: Carbamate formation (General procedure 1) between compound13 and Fmoc-VC-PAB-PNP, followed by removal of Fmoc (General procedureD), and reaction with glutaric anhydride (General procedure H). Thefinal compound was purified by reverse phase HPLC to give compound 97 asa white powder after lyophilization. MS nm/z Calcd for C₆₆H₁₀₃N₁₁O₁₇S1353.7, Found 1354.9 ([M+H]⁺).

Example IX-2. Synthesis of Compound 98

Compound 98 was synthesized using the general procedures described aboveas following: Carbamate formation (General procedure I) between compound4 and Fmoc-A-PAB-PNP, followed by removal of Fmoc (General procedure D),and amide formation with acid 87 using HATU (General procedure A, 3 eqof acid 87 and 1 eq of HATU were used). The final compound was purifiedby reverse phase HPLC to give compound 98 as a white powder afterlyophilization. MS m/z Calcd for C₆₁H₉₄N₈O₁₇S 1242.7, Found 1243.8([M+H]⁺).

Example IX-3 Synthesis of Compound 99

Compound 99 was synthesized using the general procedures described aboveas following: reaction of compound 13 and aldehyde 100 under reductivealkylation conditions (General procedure E) and Fmoc-A-PAB-PNP, followedby removal of t-Bu ester (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 99 as a white powderafter lyophilization. MS m/z Calcd for C₄₉H₈₂N₆O₁₃S 994.6, Found 995.8([M+H]⁺).

Example IX-4 Synthesis of Compound 102

Compound 102 was synthesized using the general procedures describedabove as following: Carbamate formation (General procedure I) betweencompound 16 and Fmoc-A-PAB-PNP, followed by removal of Fmoc (Generalprocedure D), and reaction with glutaric anhydride (General procedureH). The final compound was purified by reverse phase HPLC to givecompound 102 as a white powder after lyophilization. MS m/z Calcd forC₅₅H₉₀N₅O₁₅S 1134.6, Found 1135.6 ([M+H]⁺).

Example IX-5 Synthesis of Compound 103

Compound 103 was synthesized using the general procedures describedabove as following: Carbamate formation (General procedure I) betweencompound 16 and Fmoc-A-PAB-PNP, followed by removal of Fmoc (Generalprocedure D), and amide formation with acid 87 using HATU (Generalprocedure A, 3 eq of acid 87 and 1 eq of HATU were used). The finalcompound was purified by reverse phase HPLC to give compound 103 as awhite powder after lyophilization. MS m/z Calcd for C₅₈H₉₆N₈O₁₇S 1208.7,Found 1209.8 ([M+H]⁺).

Example IX-6. Synthesis of Compound 104

Compound 104 was synthesized using the general procedures describedabove as following: DIC/HOAt mediated amide bond formation (Generalprocedure B) between compound 42 and compound 66, followed by removal ofFmoc (General procedure D), and reaction with glutaric anhydride(General procedure H). The final compound was purified by reverse phaseHPLC to give compound 104 as a white powder after lyophilization. MS m/zCalcd for C₁H₁₁₅N₁₃O₁₉S 1485.8, Found 1486.9 ([M+H]⁺).

Example IX-7. Synthesis of Compound 105

Compound 105 was synthesized in the same manner as described for thesynthesis of compound 104. The final compound was purified by reversephase HPLC to give compound 105 as a white powder after lyophilization.MS m/z Calcd for C₇₃H₁₁₇N₁₃O₁₉S 1511.8, Found 1512.9 ([M+H]⁺).

Example IX-8. Synthesis of Compound 110

The phenol 106 (1 mmol) was treated with 3 eq ofbis(p-nitrophenyl)carbonate to form the activated carbonate 107 (generalprocedure G). The crude product was used directly without furtherpurification. Piperidine 4-carboxylic acid (5 eq) was dissolved in sat.aq. NaHCO₃ (5 mL) and the solution was added. The reaction mixture wasstirred at room temperature for 8 h. Citric acid (aq. 10%) was added toacidify the reaction (pH=4-5) and then diluted with EtOAc (150 mL).Organic layer was dried (over Na₂SO₄) and concentrated to give the crudeproduct 108 which underwent the following procedures: removal of Boc(General procedure C), and reductive alkylation using HCHO (Generalprocedure E). The final compound was purified by reverse phase HPLC togive compound 110 as a white powder after lyophilization. MS m/z Calcdfor C₈₀H₈₁N₇O₁₃S 1019.6, Found 1020.8 ([M+H]⁺).

Example IX-9. Synthesis of Compound 113

To a stirred solution of compound 106 (0.2 mmol, 190 mg) in anhydrousDMF (5 mL) was added t-butyl bromoacetate (0.3 mmol), followed by solidpotassium carbonate (55 mg, 0.4 mmol). The reaction mixture was stirredat room temperature for 2 h. LC/MS confirmed that the completion of thereaction. The mixture was diluted with EtOAc (100 mL) and washed with10% aq. Citric acid and brine. The organic layer was dried andconcentrated to dryness to give the crude compound 111, which underwentthe following procedures: removal of Boc and t-Bu (General procedure C),and reductive alkylation using HCHO (General procedure E). The finalcompound was purified by reverse phase HPLC to give compound 113 as awhite powder after lyophilization. MS m/z Calcd for C₄₅H₇₄N₆O₁₂S, 922.5Found 923.7 ([M+H]⁺).

Example IX-10. Synthesis of Compound 114

Compound 114 was synthesized using the general procedures describedabove as following: HATU mediated amide bond formation (Generalprocedure A) between compound 113 and methyl isonipecotate, followed bysaponification to remove methyl group from ester (General procedure F).The final compound was purified by reverse phase HPLC to give compound114 as a white powder after lyophilization. MS n/z Calcd forC₅₁H₈₃N₇O₁₃S 1033.6, Found 1034.7 ([M+H]⁺).

Example IX-11. Synthesis of Compound 115

Compound 115 was synthesized using the general procedures describedabove as following: DIC/HOAt mediated amide bond formation (Generalprocedure B) between compound 113 and compound 67, followed by removalof Fmoc (General procedure D), HATU mediated amidation reaction withacid 116 (General procedure A), and saponification to remove methylgroup from ester (General procedure F). The final compound was purifiedby reverse phase HPLC to give compound 115 as a white powder afterlyophilization. MS nm/z Calcd for C₆₅H₁₀₀N₁₀O₁₇S 1324.7, Found 1325.9([M+H]⁺).

Example X. Introduction of the Final Function Group Prior to Conjugation

MS Conjugation found ID Structure Method [M + H]⁺ 101

C 1433.9 118

C 1091.2 120

D 1829.5 133

B 1136.6 136

E 1026.6 139

F 1184.8

Example X-1. Synthesis of Compound 101

Compound 101 was synthesized using the general procedures describedabove as following: Carbamate formation (General procedure 1) betweencompound 13 and FmocVC-PAB-PNP, followed by removal of Fmoc (Generalprocedure D), and amidation reaction with 6-maleimidohexanoic acid(General procedure A). The final compound was purified by reverse phaseHPLC to give compound 101 as a white powder after lyophilization. MS m/zCalcd for C₇₁H₁₀₈N₁₂O₁₇S 1432.8, Found 1433.9 ([M+H]⁺).

Example X-2. Converting an Existing Amino Group to Maleimide DirectlyUsing N-Methoxy-Carbonylmaleimide

The amine (117, 0.1 mmol) was dissolved in acetonitrile/water (6/4, v/v,3 mL). The mixture was cooled in an ice-water bath and treated with sat.aq. NaHCO₃ (0.5 mL), followed by N-methoxycarbonylmaleimide (0.12 mmol).The mixture was stirred at room temperature for 1 h. The pH was adjustedto 6-7 with citric acid and the solution was concentrated. The residuewas purified by RP-HPLC to yield the desired maleimide 118 as a whitepowder after lyophilization (58%). MS found: 1091.2 (M+H)⁺.

Example X-3. Formation of Dibromomethyl Quinoxaline

The o-phenylenediamino compound 119 (12 mg) was dissolved inacetonitrile/water (6/4, v/v, 1 mL). NaOAc buffer (100 mM, pH=4.0, 0.3mL) was added, followed by addition of dibromomethyl diketone (10 mg).The mixture was stirred at room temperature for 10 min and purifieddirectly by RP-HPLC to give the desired quinoxaline 120 as a whitepowder (12 mg) after lyophilization. MS found 1829.5 (M+H)⁺.

Example X-4. Synthesis of Compound 136

Compound 13 (50 mg) was treated with aldehyde under reductive alkylationconditions (General procedure E). Without any purification, hydrazinehydrate (20 μL) was added to the reaction mixture. After 10 min, thecrude mixture was purified by RP-HPLC to give compound 136 as a whitepowder (46 mg). MS found 1026.6 (M+H)⁺.

Example X-5. Synthesis of Compound 139

Compound 139 was synthesized from compound 137 and carbonate 138according to General procedure I. MS found: 1184.8 (M+H)⁺.

The Antibody Drug Conjugates depicted in the Figures were preparedaccording to Example XI.

Example XI. Preparation of Antibody Drug Conjugate

To a solution of 0.5-50 mgs/mL of Trastuzumab in buffet at pH 6.0-9.0with 0-30% organic solvent, was added 0.1-10 eq of carboxylic acidactivated derivatives of compounds 84, 92, 93, or 98 in a portion wiseor continuous flow manner. The reaction was performed at 0-40° C. for0.5-50 hours with gentle stirring or shaking, monitored by HIC-HPLC(Hydrophobic Interaction Chromatography-HPLC). The resultant crude ADCproduct underwent necessary down-stream steps of desalt, buffetchanges/formulation, and optionally, purification, using thestate-of-art procedures. The final ADC product was characterized byHIC-HPLC, SEC, RP-HPLC, and optionally LC-MS. The average DAR (drugantibody ratio) was calculated by UV absorption and/or MS spectroscopy.

Example XII. In Vitro Cytotoxicity Experiment

The cell lines used were SK-BR-3 human breast adenocarcinoma (HER2triple positive), HCC1954 human Ductal Carcinoma (HER2 triple positive),MCF7 human breast adenocarcinoma (HER2 normal), and MDA-MB-468 humanbreast adenocarcinoma (HER2 negative). These cells were available fromATCC. SK-BR-3 cells were grown in McCoy's 5A medium (Caisson Labs, NorthLogan, Utah) supplemented with 10% fetal bovine serum. HCC1954 cellswere grown in RPMI-1640 medium (Caisson Labs, North Logan, Utah)supplemented with 10% fetal bovine serum. MCF7 and MDA-MB-468 cells weregrown in DMEM/F12 medium (Caisson Labs, North Logan, Utah) supplementedwith 10% fetal bovine serum. SK-BR-3, MCF7, and MDA-MB-468 cells wereplated in 96-well plates at approximately 7,500 cells/well, and HCC1954cells were plated in 96-well plates at approximately 20,000 cells/well.Compounds or the antibody-drug conjugates were added in duplicates inthe same day. After 72 hour incubation at 37° C., CellTiter-Glo(Promega, Madison, Wis.) were added and cell viability was determined asdescribe by the manufacture's protocol. The percent viability wasdetermined as following:

% Viability=Average Luminescence Value of the duplicates (treatedwells)/Average Luminescence Value of the untreated wells

1. A compound having the structure of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: A is a tubulinbinding moiety; B is an functional moiety; and R¹-R⁸ are eachindependently selected from the group consisting of H (hydrogen),optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted aryl, and optionally substitutedheteroaryl, or optionally R¹ and R² together with the nitrogen to whichthey are attached are an optionally substituted 5- to 7-membered ring,or optionally R¹ and R³ together with the atoms to which they areattached are an optionally substituted 5- to 7-membered ring, oroptionally R⁷ and Re together with the atoms to which they are attachedare an optionally substituted 5- to 7-membered ring, or optionally R¹ isR^(1A) or R^(1B); R^(1A) comprises a targeting moiety; R^(1B) is-L¹(CH₂)_(n)R^(C), -L¹O(CH₂)_(n)R^(C) or —(CH₂)_(n)R; R^(C) is C₁-C₈alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, or heterocyclyl, eachoptionally substituted with one or more R^(D), or optionally R^(C)comprises a targeting moiety; each R^(n) is independently selected fromthe group consisting of —OH, —N₃, halo, cyano, nitro,—(CH₂)_(n)NR^(E)R^(F), —(CH₂)_(n)C(═O)NR^(E)R^(F),—O(CH₂)_(n)NR^(E)R^(F), —O(CH₂)_(n)C(═O)NR^(E)R^(F),—O(CH₂)_(m)OC(═O)NR^(E)R^(F), —NR^(G)C(═O)R^(H), —NR^(G)S(O)R^(H),—O(CH₂)_(m)O(CH₂)_(m)R^(J), —O(CH₂)_(n)C(═O)R^(J), —O(CH₂)_(n)R^(J),optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted heterocyclyl, and optionallysubstituted —O(C₁-C₈ alkyl); R^(E) and R^(F) are each independentlyselected from hydrogen,-[(L¹)_(s)(C(R^(2A))₂)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-[L¹(C(R^(2A))₂)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]_(s)-(L¹)_(s)-R^(J),-[(L¹(C(R^(A))₂)(NR^(2A))_(s)(C(R^(2A))₂)_(r)]-(L¹)[(C(R^(2A))₂)_(r)O(C(R^(2A))₂)_(r)(L²)_(s)]_(s)-(L¹)_(s)-R^(J),optionally substituted C₁₋₈ alkyl, optionally substituted C₃₋₈cycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted heterocyclyl; each R^(G) isindependently hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted C₃-C₈ cycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, or optionally substituted heterocyclyl; eachR^(H) is independently hydrogen, optionally substituted C₁-C₈ alkyl,optionally substituted C₃-C₈ cycloalkyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclyl,or —NR^(E)R^(F); each R is independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈ cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, —(CH₂)_(n)OR^(2B),—O(CH₂)_(n)OR^(2B), —(CH₂)_(n)NR^(2B)R^(2B), —C(R^(2A))₂NR^(2B)R^(2B),—(CH₂)_(n)C(═O)OR^(2B), and —C(═O)NHR^(2B); each R^(2A) is independentlyselected, wherein R^(2A) is selected from the group consisting ofhydrogen, halo, —OH, optionally substituted C₁-C₃ alkyl, optionallysubstituted —O—(C₁-C₈ alkyl), optionally substituted C₃-C₈ cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, —(CH₂)_(n)OR^(2B),—(CH₂)_(n)NR^(2C)R^(2C), —C(═O)OR^(2B), and —C(═O)NR^(2C)R^(2C), oroptionally two geminal R^(2A) and the carbon to which they are attachedform an optionally substituted three- to six-membered carbocyclic ring;each R^(2B) is independently selected from the group consisting ofhydrogen, OH, —(CH₂)_(n)C(═O)OH, —C(═O)(C(R^(2D))₂)_(n)L³R^(2E),optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted —O—(C₁-C₈ alkyl), optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocyclyl; each R^(2c) is independently selected from thegroup consisting of hydrogen, —OH, optionally substituted C₁-C₈ alkyl,optionally substituted C₃-C₈ cycloalkyl, optionally substituted—O—(C₁-C₈ alkyl), optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted heterocyclyl, or optionally bothR^(2c) together with the nitrogen to which they are attached are anoptionally substituted heterocyclyl; each R^(2D) is independentlyselected from the group consisting of hydrogen, optionally substitutedC₁-C₈ alkyl, optionally substituted C₁-C₈ cycloalkyl, optionallysubstituted —O—(C₁-C₈ alkyl), optionally substituted aryl, optionallysubstituted heteroaryl, and optionally substituted heterocyclyl; eachR_(2E) is independently selected from the group consisting of optionallysubstituted C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl, andoptionally substituted heterocyclyl, and —(CH₂)_(n)C(═O)OR^(2F); eachR^(2F) is independently selected from the group consisting hydrogen,optionally substituted C1-C8 alkyl, optionally substituted C3-C8cycloalkyl, optionally substituted aryl, optionally substitutedheteroaryl, and optionally substituted heterocyclyl; each L¹ isindependently selected from the group consisting of —C(═O)—, —S(═O)—,—C(═S)—, —S(═O)₂—, —C(═O)O—, —C(═O)NR^(2A)—, —S(═O)NR^(2A)—,—S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and —C(CF₃)₂NR^(2A)—; each L² isindependently selected from the group consisting of optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted heterocyclyl; each L³ is independently selected from thegroup consisting of —C(═O)—, —S(═O)—, —C(═S)—, —S(═O)₂—, —C(═O)O—,—C(═O)NR²—, —S(═O)NR^(2A)—, —S(═O)₂NR^(2A)—, —C(═O)NR^(2A)C(═O)—, and—C(CF₃)₂NR^(2A)—; each m independently is 1 or 2; each n independentlyis 0, 1, 2, 3, 4, 5, or 6; each r independently is 0, 1, 2, 3, 4, 5, or6; each s independently is 0 or 1; each z independently is 1 or 2; R⁷ isselected from the group consisting of H (hydrogen), optionallysubstituted C₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl,optionally substituted aryl, and optionally substituted heterocyclyl; R⁸is selected from the group consisting of H (hydrogen), —(CH₂)_(n)R^(C),optionally substituted C₁-C₈ alkyl, optionally substituted C₃-C₈cycloalkyl, optionally substituted aryl, and optionally substitutedheterocyclyl; and R is selected from the group consisting of H(hydrogen), optionally substituted C₁-C₈ alkyl, optionally substitutedC₃-C₈ cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl. 2-60. (canceled)